Targeting tissue factor to activated platelets

ABSTRACT

The current invention relates to procoagulant fusion proteins, polynucleotides that encode said fusion proteins and cells that expresses said fusion proteins. Furthermore, the current invention relates to fusion proteins for use as a medicament. Individuals that have a coagulopathy, such as haemophilia A and B with or without inhibitors, may be treated with fusions proteins of the current invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/090,002, Apr. 4, 2016, which is a continuation of U.S. applicationSer. No. 13/391,755 filed on Mar. 22, 2012 (now abandoned), which is a35 U.S.C. § 371 national stage application of International PatentApplication PCT/EP2010/062519 (published as WO 2011/023785 A1), filedAug. 26, 2010, which claimed priority of European Patent Application09168833.3, filed Aug. 27, 2009; this application further claimspriority under 35 U.S.C. § 119 of U.S. Provisional Application61/239,142, filed Sep. 2, 2009 and U.S. Provisional Application61/288,944, filed Dec. 22, 2009; the contents of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The current invention relates to procoagulant fusion proteins, apolynucleotides that encode said procoagulant fusion proteins, cellsthat expresses said procoagulant fusion protein and uses thereof.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 11, 2018, isnamed 8076US05_SeqList.txt and is 154 kilobytes in size.

BACKGROUND OF THE INVENTION

Tissue factor (TF), a transmembrane glycoprotein, is the primarycellular initiator of blood coagulation. It is predominantly expressedon the surface of sub-endothelial cells, such as smooth muscle cells andfibroblasts, and binds both zymogen coagulation Factor VII (FVII) andthe activated form, Factor VIIa (FVIIa) when the integrity of theendothelium is interrupted, such as when blood vessels are severed. WhenTF binds FVII, it promotes FVII to FVIIa activation. TF also greatlyenhances the proteolytic activity of Factor VIIa towards its physiologicsubstrates, Factors IX and X. FVIIa retains a zymogen-like state insolution, acting as a relatively poor enzyme. TF provides a scaffold foroptimal macromolecular exosite interaction, induces conformationalchanges in the protease domain of FVIIa leading to maturation of itsactive site and orientates FVIIa on a cell surface for optimal substrateinteraction. Together these effects results in an enhanced catalyticcapability of FVIIa of several orders of magnitude. Hence, TF is aco-factor for FVIIa in the initiation complex of what is traditionallyreferred to as the extrinsic pathway of blood coagulation. Subsequentsteps of the coagulation cascade finally result in the formation of afibrin polymer which is bound by activated platelets and cross-linkedwith FXIIIa.

Platelets—also known as thrombocytes—derive from their cellularpredecessor, megakaryocytes. Normal resting platelets freely flowthroughout the blood circulation when the endothelium is intact. Whenthe single-layered endothelial barrier is damaged, resting plateletsadhere to subendothelial structures by means of glycoprotein (GP)receptors. For example, GPIaIIa and GPVI bind collagen; GPIcIIa bindsfibronectin; GPIc*IIa binds laminin and GPIb-V-IX binds von WillebrandFactor (vWF) polymers. Adhesion of platelets in this manner causes themto change shape and release their alpha and dense granules. In turn,this results in the exposure of a plethora of other glycoproteinplatelet receptors, such as GPIIbIIIa (which binds fibrinogen/fibrin)and TREM-like transcript 1 (TLT-1); as well as the release ofcoagulation factors I (fibrinogen), V and XI; other procoagulants suchas ADP, Ca²⁺, serotonin and Platelet Factors 3/4; anti-coagulants suchas tissue factor pathway inhibitor (TFPI); and compounds such asplatelet derived growth factor (PDGF), essential to plateletreplenishment and healing. Activated platelets bind to one other andcross-link fibrin in a rapid reaction for example via the GPIIbIIIareceptor complex.

Hence, activated platelets and the fibrin polymer product of thecoagulation cascade together form the blood clot. Platelet aggregationof coagulation is known as the primary hemostatic response, while thecoagulation cascade response is known as the secondary hemostaticresponse. Although fibrin is produced during the primary hemostaticresponse, via the so-called coagulation initiation (independent ofFIX/FVIIIa), the amount produced at this point is insufficient for astrong coagel. Initial fibrin serves as an aggregater of activatedplatelets at site of injury, which again provides an optimal cellsurface for the function of activated coagulation factors.

In subjects with a coagulopathy, such as in human beings withhaemophilia A and B, various steps of the coagulation cascade arerendered dysfunctional due to, for example, the absence or insufficientpresence of a coagulation factor. Such dysfunction of one part ofcoagulation results in insufficient blood coagulation and potentiallylife-threatening bleeding.

An object of the current invention is to provide a compound that issuitable for use as a procoagulant drug in such subjects. A secondobject of the current invention is to provide a compound that enables aphysical point of initiation of blood coagulation to be mobilised, suchthat extrinsic coagulation is not solely dependent on subendothelial,cell-bound tissue factor. A third object of the current invention is toprovide a compound that up-regulates blood coagulation in aphysiologically suitable microenvironment. A further object of thecurrent invention is to direct a soluble tissue factor, or abiologically functional fragment or variant thereof, to the surface ofactivated platelets. A further object of the invention is to enhance theproteolytic activity of endogenous Factor VIIa towards its physiologicalsubstrates, Factors IX and X. Thus, the object is to enable theinitiation of blood coagulation on the surface of activated plateletsthat are located intravascularly or extravascularly. This is in additionto the normal and exclusively subendothelial—typicallyextravascular—initiation of blood coagulation.

WO06/096828 discloses chimeric proteins that comprise soluble tissuefactor (sTF) and a phosphatidyl serine (PS) binding domain, such asAnnexin V. PS is exposed on the surface of activated cells, such asmonocytes, endothelial cells and cells undergoing apoptosis, as well ason activated and resting platelets. The chimeric proteins are bothpro-coagulant and anti-coagulant; the latter due to the fact that, inhigher doses, constructs compete with coagulation factors in binding toPS on activated platelets. Thus, the chimeric proteins of WO06/096828have a different set of properties than the fusion proteins describedherein.

SUMMARY OF THE INVENTION

The current invention provides a fusion protein comprising (i) at leastone tissue factor polypeptide, or biologically functional variant(s) orfragment(s) thereof, which is/are covalently attached to (ii) a ligandthat is capable of binding (iii) a receptor, and/or a fragment thereof,wherein the receptor is only expressed on the surface of activatedplatelets. The fusion protein/construct may comprise (i) tissue factor,or a biologically functional variant or fragment thereof, and (ii) aligand that binds (iii) TLT-1. According to the current invention, (ii)may be a monoclonal antibody, or an antigen-binding portion of amonoclonal antibody. For example, (ii) may be selected from the groupconsisting of: a Fab fragment, a F(ab′)₂ fragment, a Fab′ fragment, a Fdfragment, a Fv fragment, a dAb fragment or an isolated complementaritydetermining region (CDR). According to the current invention, (ii) maycomprise the variable domain of 0012 (2F105) LC. According to thecurrent invention, (ii) may comprise the variable domain of 0012(otherwise referred to as 2F105) LC together with the constant region ofhuman LC, kappa and a HPC4 tag (pTT-0012LC.HPC4, also referred to aspTT-2F105LC.HPC4).

The fusion proteins/constructs of the present invention target theinitial stages of platelet-clot growth through the specific targeting ofactivated platelets, while concurrently recruiting resting or basalplatelets directly to the site of injury, thereby activating saidresting or basal platelets systemically. The compositions of the presentinvention are based upon the identification of particular receptors andcomponent epitopes, that appear on platelet membranes when platelets areno longer resting but in the process of being activated.

The current invention also provides the following: an isolatednucleotide sequence that encodes any fusion protein/construct accordingto the current invention; a vector that comprises an isolated nucleotidesequence that, in turn, encodes any fusion protein/construct accordingto the current invention; an isolated cell that comprises a nucleotidesequence that encodes any fusion protein/construct of the currentinvention. Said nucleotide sequence may, in turn, be expressed by anintracellular vector. Said isolated cell may be a eukaryotic cell, suchas a mammalian cell, such as a BHK or a CHO or a HEK cell.

Furthermore, the current invention provides a method of targeting tissuefactor to the surface of activated platelets, said method comprisingcontacting activated platelets with any construct comprising (i) tissuefactor, or a functional variant thereof, and (ii) a ligand that iscapable of binding (iii) a receptor present on an activated platelet,such as TLT-1. In this way, the invention also relates to a method ofup-regulating FX activation on the surface of activated platelets,wherein said method comprises the contacting of activated platelets withsaid construct in the simultaneous presence of FX.

Similarly, the invention relates to fusion proteins for use as amedicament and for use in the treatment of a coagulopathy. In oneembodiment, a therapeutically effective amount of said construct isparenterally administered, such as intravenously or subcutaneouslyadministered, to an individual in need thereof. Such individual in needmay have any congenital, acquired and/or iatrogenic coagulopathy.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Human TLT-1 nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO:2) sequences. In FIG. 1, the nucleotide- and amino acid sequencesrepresenting hTLT-1 are shown. Here, nucleotide sequence position 1-45encode the predicted signal peptide, the nucleotide sequence at position46-486 encode the extracellular domain of hTLT-1, the nucleotidesequence position 487-555 encode the transmembrane region and thenucleotide sequence at position 556-933 encode the intracellular domainof hTLT-1.

FIG. 2: Nucleotide (SEQ ID NO: 3) and amino acid (SEQ ID NO: 4)sequences representing the extracellular domain of human TLT-1containing a C-terminal His-6 tag. In FIG. 2, the nucleotide- and aminoacid sequences representing the extracellular domain of hTLT-1 with aC-terminal His tag are shown. Here, the underlined sequence (7-15)indicates the positions of a kozak sequence, the nucleotide sequence atposition 16-60 encodes the predicted signal peptide, the nucleotidesequence at position 61-501 encode the extracellular domain of hTLT-1and the nucleotide sequence at position 502-519 encodes the 6×His tag(in bold). The restriction enzyme sites HindIII (nucleotide sequence atposition 1-6) and EcoRI (nucleotide sequence at position 523-528) arealso shown and the stop codon is marked with an asterisk (520-522).

FIGS. 3A-3D: The variable domain of 0012LC and 0012HC including Kabatnumbering:

FIG. 3A shows variable domain of 0012LC

FIG. 3B shows variable domain of 0012LC—Kabat numbering

FIG. 3C shows variable domain of 0012HC

FIG. 3D shows variable domain of 0012HC—Kabat numbering.

In FIG. 3A, the nucleotide sequence (SEQ ID NO: 9) at position 1-57encodes the LC signal peptide (SEQ ID NO: 10) sequence; the nucleotidesequences at position 58-396 encodes the variable domain of 0012LC. InFIG. 3B (SEQ ID NO: 164), the sequences in bold and grey representpositions of the 0012LC CDRs according to Kabat numbering. In FIG. 3C(SEQ ID NO: 11 and SEQ ID NO: 12), the nucleotide sequence at position1-54 encodes the 0012HC signal peptide, the nucleotide sequence atposition 55-396 encodes the variable domain of 0012HC. In FIG. 3D (SEQID NO: 165), the sequences in bold and grey represent positions of the0012HC CDRs according to Kabat numbering.

FIG. 4: The variable domain of 0012LC together with constant region ofhuman LC,kappa and a HPC4 tag (encoded by pTT-0012LC.HPC4) (SEQ ID NO:166 and SEQ ID NO: 167). In FIG. 4, the nucleotide sequences at position1-57 encodes the LC signal peptide, the nucleotide sequence 58-396encodes the variable domain of 0012LC, the nucleotide sequence 397-714encodes the constant region of human 0012LC, kappa and the nucleotidesequence 715-750 encodes a HPC4 tag.

FIG. 5: The variable domain of 0012HC together with the constant regionof human IgG4 (pTT-0012HC) (SEQ ID NO: 17 and SEQ ID NO: 39). In FIG. 5the nucleotide sequence 1-54 encodes the HC signal peptide, thenucleotide sequence 55-396 encodes the variable domain of 0012HC and thenucleotide sequence 397-1377 encodes the constant region of human IgG4(in bold).

FIG. 6: The nucleotide- (SEQ ID NO: 169) and amino acid (SEQ ID NO: 168)sequences of 0012V_(H)-CH1-L4a-hTF.1-219, as encoded bypTT-0012V_(H)-CH1-L4a-hTF.1-219. The pTT-0012V_(H)-CH1-L4a-hTF.1-219construct encoding the Gly-Ser linker L4a and the extracellular domainAA 1-219 of human tissue factor. Here, the nucleotide sequence atposition 1-54 encodes the predicted signal peptide, the nucleotidesequence at position 55-396 encode the variable domain of 0012HC, thenucleotide sequence at position 397-699 (in bold) encode the human IgG4CH₁ constant region, the nucleotide sequence at position 700-750(underlined) encodes the Gly-Ser linker designated L4a and thenucleotide sequence at position 701-1407 encodes the extracellulardomain of human tissue factor (amino acid 1-219, dotted sequence). The0012V_(H)-CH1-L4a-hTF.1-219 amino acid sequence is composed of SEQ IDno. 51 (0012VH-VH1), SEQ ID no. 61 (L4a) and SEQ ID no. 14 (hTF.1-219).

FIG. 7: Stimulation of factor VIIa activity. The data in FIG. 7demonstrate that a similar concentration-dependent stimulation of FVIIaamidolytic activity was obtained with Fab-hTF.1-219 and mAb-hTF.1-219fusion proteins as with non-fused hTF.1-219. This indicates that bindingof FVIIa to the TF component of TF-fusion proteins was not affected bythe linking of TF to Fab or mAb fragments.

FIGS. 8A-8B: Binding of protein ID no. 0010 and 0011 to activatedplatelets by FACS analysis. Detection via an anti-HPC4 tag (FIG. 8A) oran anti-LC antibody (FIG. 8B).

FIG. 9: FIG. 9A shows the effect of i) 0.03 nM INNOVIN® (lipidatedtissue factor) ii) 10 nM sTF, iii) 10 nM 0011 Fab-hTF.1-219, iv) 10 nM0010 Fab anti-TLT-1 antibody or v) 10 nM 0012 mAb anti-TLT-1 antibody onFVIIa-mediated FX activation in the absence or presence of platelets.FIG. 9B shows the effect of replacing rFVIIa in the assay with thezymogen, rFVII.

FIGS. 10A-10B: The data in FIG. 10A shows that binding of 10 nM 0011Fab-hTF.1-219 to the TLT-1 receptor specifically stimulatesFVIIa-mediated FX activation on activated and not on resting platelets.Stimulation increases with the number of activated platelets and issaturated with an EC₅₀≈12.000 plts/μl. FIG. 10B compares the results inFIG. 10A with the results obtained with 0.1 nM Annexin V-hTF.1-219fusion protein (AV-hTF.1-219). FIG. 10B shows that binding ofAV-hTF.1-219 to platelet phospholipid stimulates FX activation on bothactivated and resting platelets. A marked stimulation of FX activationis observed with resting platelets which at the maximal platelet numbertested exceeds FXa generation on the surface of an equivalent number ofactivated platelets. FIG. 10B clearly shows that AV-TF cannot be used toselectively enhance FXa generation on activated platelets.

FIG. 11: The data in FIG. 11 shows the FVIIa/FVII concentrationdependency of the stimulation of FX activation on activated platelets by10 nM of Fab-hTF.1-219 fusion proteins. The effect with activatedplatelets of 0020 Fab-hTF.1-219 is compared to the effect of a 0094Fab-hTF.1-219 isotype fusion protein which do not bind TLT-1 and tonon-fused hTF.1-219. Also shown for comparison is the effect of 0020Fab-hTF.1-219 with resting platelets. A similar stimulation is obtainedat all concentrations with FVIIa and FVII showing that TF-fusionproteins mediate an efficient feed-back activation of FVII on activatedplatelets and that stimulation is optimal at physiologicalconcentrations of FVII/FVIIa. A marked stimulation is induced upon TLT-1targeting and not with non-targeting hTF.1-219 derivatives.

FIG. 12: The data in FIG. 12 shows the stimulation of FX activationmediated by 10 nM FVIIa/FVII on activated platelets at variousconcentrations of Fab-hTF.1-219 fusion proteins. The effect withactivated platelets of 0070 Fab-hTF.1-219 is compared to the effect of a0094 Fab-hTF.1-219 isotype fusion protein which do not bind TLT-1 and tonon-fused hTF.1-219. The 0070 Fab-hTF.1-219 markedly stimulates FXactivation over a wide concentration range with a mechanism whichrequires targeting to TLT-1 as indicated by comparison with non-bindingcontrols.

FIG. 13: The data in FIG. 13 shows the stimulation of FX activationmediated by o.1 nM FVIIa/FVII on TLT-1 enriched phospholipids at variousconcentrations of Fab-hTF.1-219 fusion proteins. The effect of 0070Fab-hTF.1-219 is compared to the effect of a 0094 Fab-hTF.1-219 isotypefusion protein which do not bind TLT-1 and to non-fused hTF.1-219. The0070 Fab-hTF.1-219 markedly stimulates FX activation over a wideconcentration range with a mechanism which requires targeting to TLT-1as indicated by comparison with non-binding controls.

FIGS. 14A-14B: FIGS. 14A-14B show the stimulation of fibrin clotformation in “hemophilia like” human whole blood (HWB) mediated by 0070Fab-hTF.1-219 fusion protein. FIG. 14A shows TEG traces of clotformation measured at various concentrations (0, 0.1, 0.2, 1.0, and 10nM) of 0070 Fab-hTF.1-219 fusion protein added to HWB made “hemophilialike” by addition of an antibody against human FVIII. The clotting timeis markedly reduced by the FVIII antibody and increasing concentrationsof 0070 Fab-hTF.1-219 dramatically revert the effect of the FVIIIantibody. FIG. 14B shows the R times for the TEG traces in FIG. 14A andcompares the R time for 0070 Fab-hTF.1-219 to the R times obtained forthe 0094 Fab-hTF.1-219 isotype fusion protein and hTF.1-219 run inparallel with the same donor. The 0070 Fab-hTF.1-219 markedly stimulatesclot formation in “hemophilia like” HWB with a mechanism which requirestargeting to TLT-1 as indicated by comparison with non-binding controls.

FIG. 15: shows that TLT1-FAb-TF reduces the tail bleeding blood loss inhaemophilic mice transfused with human platelets 2 min before inductionof bleeding, when compared to an irrelevant FAb-TF-construct.

FIG. 16: Sequence coverage of HX analyzed peptides of TLT-1 in thepresence and absence of 0023. The primary sequence (using maturenumbering) (SEQ ID NO: 170) is displayed above the HX analyzed peptides(shown as horizontal bars). Peptides showing similar exchange patternsboth in the presence and absence of 0023 are displayed in white whereaspeptides showing reduced deuterium incorporation upon 0023 binding arecoloured black.

FIG. 17: Sequence coverage of HX analyzed peptides of TLT-1 in thepresence and absence of 0051. The primary sequence (using maturenumbering) (SEQ ID NO: 170) is displayed above the HX analyzed peptides(shown as horizontal bars). Peptides showing similar exchange patternsboth in the presence and absence of 0051 are displayed in white whereaspeptides showing reduced deuterium incorporation upon 0051 binding arecoloured black.

FIG. 18: Sequence coverage of HX analyzed peptides of TLT-1 in thepresence and absence of 0062. The primary sequence (using maturenumbering) (SEQ ID NO: 170) is displayed above the HX analyzed peptides(shown as horizontal bars). Peptides showing similar exchange patternsboth in the presence and absence of 0062 are displayed in white whereaspeptides showing reduced deuterium incorporation upon 0062 binding arecoloured black.

FIG. 19: Sequence coverage of HX analyzed peptides of TLT-1 in thepresence and absence of 0061. The primary sequence (using maturenumbering) (SEQ ID NO: 171) is displayed above the HX analyzed peptides(shown as horizontal bars). Peptides showing similar exchange patternsboth in the presence and absence of 0061 are displayed in white whereaspeptides showing reduced deuterium incorporation upon 0061 binding arecoloured black.

FIG. 20: Sequence coverage of HX analyzed peptides of TLT-1 region126-162. The primary sequence (using mature numbering) (SEQ ID NO: 172)is displayed above the HX analyzed peptides (shown as horizontal bars).All peptides showed reduced deuterium incorporation upon 0061 binding.

FIG. 21: Plasmid map for the expression vector pTT. DNA fragments can beinserted into the HindIII and EcoRI restriction enzyme sites.

FIG. 22: Plasmid map for the expression vector pTT-hIgG4. The expressionvector contains the human IgG4 CH1-hinge-CH2-CH3 DNA sequences. V_(H)encoding DNA sequences can be inserted into the HinDIII and NheIrestriction enzyme sites resulting in a complete HC encoding plasmid.

FIG. 23: Plasmid map for the expression vector pTT-hCL,kappa. Theexpression vector contains the DNA sequences encoding the constantregion of human LC,kappa designated hCL,kappa. V_(L) encoding DNAsequences can be inserted into the HinDIII and BslWI restriction enzymesites resulting in a complete LC encoding plasmid.

FIG. 24: Plasmid map for the expression vector pTT-L4a-hTF.1-219. Theexpression vector contains DNA sequences encoding a 17 amino acid longGly-Ser linker and human TF.1-219. HC, LC or V_(H)—CH1 encoding DNAsequences can be inserted into the HinDIII and BamHI restriction enzymesites resulting in HC-L4a-hTF.1-219, LC-L4a-hTF.1-219 orV_(H)—CH1-L4a-hTF.1-219 encoding plasmids.

FIG. 25: Plasmid map for the expression vector pTT-hTF.1-219-L4b. Theexpression vector contains DNA sequences encoding human TF.1-219 and a17 amino acid long Gly-Ser linker. HC, LC or V_(H)—CH1 encoding DNAsequences can be inserted into the BamHI and EcoRI restriction enzymesites resulting in hTF.1-219-L4b-HC, hTF.1-219-L4b-LC orhTF.1-219-L4b-V_(H)-CH1 encoding plasmid.

FIG. 26: Scheme of mAb-LC-hTF.1-219 and Fab-LC-hTF.1-219 constructs.Standard denotations for antibody components are shown: VL, CL, VH, CH1,CH2, and CH3. Tissue factor's fibronectin III domains are denotedFbn-III.

FIGS. 27A-27B: Screening of TF-fusion protein pro-coagulant activity bymeasuring FVII/FVIIa-mediated activation of FX on i) activated plateletsand on ii) TLT-1 enriched phospholipids. Stimulation of FX activation onactivated platelets was performed as described in Example 37 onplatelets from individual human donors. The stimulation obtained with0020 Fab-hTF.1-219 was always included and set to 100%. Stimulation ofFX activation TLT-1 enriched phospholipids was performed as described inExample 40 with 0.1 nM FVII/FVIIa and 10 nM Fab fusion protein or 1.0 nMmAB fusion protein. The stimulation obtained with 0020 Fab-hTF.1-219 wasalways included and set to 100%. FIG. 27A lists data on mAb-TF fusionproteins and FIG. 27B lists data on Fab-TF fusion proteins. Also showare data with 0020 Fab-hTF.1-219 on resting platelets and results withnon-binding isotype antibodies.

SEQUENCES

The sequences are as follows:

SEQ ID NO: 1 provides the nucleotide sequence of human (h)TLT-1.

SEQ ID NO: 2 provides the amino acid sequence of hTLT-1.

SEQ ID NO: 3 provides the nucleotide sequence of the extracellulardomain of hTLT-1-His6.

SEQ ID NO: 4 provides the amino acid sequence of the extracellulardomain of hTLT-1-His6.

SEQ ID NOs: 5 to 8 provide the amino acid sequences of hTLT-1 fragments:hTLT-1.20-125, hTLT-1.16-162, hTLT-1.126-162 and hTLT-1.129-142.

SEQ ID NO: 9 provides the nucleotide sequence of the variable domain ofmAb 0012 (2F105), LC.

SEQ ID NO: 10 provides the amino acid sequence of the variable domain ofmAb 0012 (2F105), LC.

SEQ ID NO: 11 provides the nucleotide sequence of the variable domain of0012 (2F105) HC.

SEQ ID NO: 12 provides the amino acid sequence of the variable domain of0012 (2F105) HC.

SEQ ID NO: 13 provides the nucleic acid sequence of hTF.1-219.

SEQ ID NO: 14 provides the amino acid sequence of hTF.1-219.

SEQ ID NO: 15 provides the nucleic acid sequence of human tissue factor.

SEQ ID NO: 16 provides the amino acid sequence of human tissue factor.

SEQ ID NO: 17 provides the nucleotide sequence of the heavy chain of mAb0012.

SEQ ID NO: 18 provides the nucleotide sequence of the light chain of mAb0012 and Fab 0012.

SEQ ID NO: 19 provides the nucleotide sequence of the heavy chain of mAb0023.

SEQ ID NO: 20 provides the nucleotide sequence of the light chain of mAb0023 and Fab 0023.

SEQ ID NO: 21 provides the nucleotide sequence of the heavy chain of mAb0051.

SEQ ID NO: 22 provides the nucleotide sequence of the light chain of mAb0051 and Fab 0051.

SEQ ID NO: 23 provides the nucleotide sequence of the heavy chain of mAb0052.

SEQ ID NO: 24 provides the nucleotide sequence of the heavy chain of mAb0062.

SEQ ID NO: 25 provides the nucleotide sequence of the light chain of mAb0052, Fab 0052 and mAb 0062.

SEQ ID NO: 26 provides the nucleotide sequence of the heavy chain of mAb0061.

SEQ ID NO: 27 provides the nucleotide sequence of the heavy chain of mAb0082.

SEQ ID NO: 28 provides the nucleotide sequence of the light chain of mAb0061, Fab 0061, mAb 0082 and Fab 0082.

SEQ ID NO: 29 provides the nucleotide sequence of Fab 0012 VH-CH1.

SEQ ID NO: 30 provides the nucleotide sequence of Fab 0023 VH-CH1.

SEQ ID NO: 31 provides the nucleotide sequence of Fab 0051 VH-CH1.

SEQ ID NO: 32 provides the nucleotide sequence of Fab 0052 VH-CH1.

SEQ ID NO: 33 provides the nucleotide sequence of Fab 0061 VH-CH1.

SEQ ID NO: 34 provides the nucleotide sequence of Fab 0082 VH-CH1.

SEQ ID NO: 35 provides the nucleotide sequence of Fab AP-3 VH-VH1.

SEQ ID NO: 36 provides the nucleotide sequence of Fab AP-3 LC.

SEQ ID NO: 37 provides the nucleotide sequence of Fab-AP-3 LC.C34S.

SEQ ID NO: 38 provides the nucleotide sequence of hIgG4 hinge-CH2-CH3.

SEQ ID NO: 39 provides the amino acid sequence of mAb 0012, HC (mouseVH-human IgG4 CH1-CH2-CH3).

SEQ ID NO: 40 provides the amino acid sequence of mAb 0012, LC (mouseVL-human Kappa CL) and Fab 0012, LC (mouse VL-human Kappa CL).

SEQ ID NO: 41 provides the amino acid sequence of mAb 0023, HC (mouseVH-human IgG4 CH1-CH2-CH3).

SEQ ID NO: 42 provides the amino acid sequence of mAb 0023, LC (mouseVL-human Kappa CL) and Fab 0023, LC (mouse VL-human Kappa CL).

SEQ ID NO: 43 provides the amino acid sequence of mAb 0051, HC (mouseVH-human IgG4 CH1-CH2-CH3).

SEQ ID NO: 44 provides the amino acid sequence of mAb 0051, LC (mouseVL-human Kappa CL) and Fab 0051, LC (mouse VL-human Kappa CL).

SEQ ID NO: 45 provides the amino acid sequence of mAb 0052, HC (mouseVH-human IgG4 CH1-CH2-CH3).

SEQ ID NO: 46 provides the amino acid sequence of mAb 0052, LC (mouseVL-human Kappa CL); Fab 0052, LC (mouse VL-human Kappa CL); mAb 0062, LC(mouse VL-human Kappa CL).

SEQ ID NO: 47 provides the amino acid sequence of mAb 0061, HC (mouseVH-human IgG4 CH1-CH2-CH3).

SEQ ID NO: 48 provides the amino acid sequence of mAb 0061, LC (mouseVL-human Kappa CL); Fab 0061, LC (mouse VL-human Kappa CL) and mAb 0082,LC (mouse VL-human Kappa CL); Fab 0082, LC (mouse VL-human Kappa CL).

SEQ ID NO: 49 provides the amino acid sequence of mAb 0062, HC (mouseVH-human IgG4 CH1-CH2-CH3).

SEQ ID NO: 50 provides the amino acid sequence of mAb 0082, HC (mouseVH-human IgG4 CH1-CH2-CH3).

SEQ ID NO: 51 provides the amino acid sequence of Fab 0012, mouseVH-human IgG4 CH1.

SEQ ID NO: 52 provides the amino acid sequence of Fab 0023, mouseVH-human IgG4 CH1.

SEQ ID NO: 53 provides the amino acid sequence of Fab 0051, mouseVH-human IgG4 CH1.

SEQ ID NO: 54 provides the amino acid sequence of Fab 0052, mouseVH-human IgG4 CH1.

SEQ ID NO: 55 provides the amino acid sequence of Fab 0082, mouseVH-human IgG4 CH1.

SEQ ID NO: 56 provides the amino acid sequence of Fab AP-3, mouseVH-human IgG4 CH1.

SEQ ID NO: 57 provides the amino acid sequence of Fab AP-3, LC (mouseVL-human Kappa CL).

SEQ ID NO: 58 provides the amino acid sequence of Fab AP-3.LC.C34S, LC(mouse VL-human Kappa CL).

SEQ ID NOs: 59-68 provide the amino acid sequences of optional linkersL2-L10. Optional linkers are numbered and listed in Table 1.

SEQ ID NO: 69 provides the amino acid sequence of purification tag HPC4.

SEQ ID NOs: 70-155 provide the nucleic acid sequences of the primersused during the development of the expression constructs described inexamples 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 24,25.

SEQ ID NO: 156 provides the amino acid sequence of Fab 0061 VH-CH1.

SEQ ID NO: 157 provides the amino acid sequence of hIgG4-hinge-CH2-CH3.

SEQ ID NO: 158 provides the amino acid sequence of a His6 tag.

SEQ ID NO: 159 provides the amino acid sequence of hTLT-1.18-188.

SEQ ID NO: 160 provides the nucleic acid sequence of primer no. 1004.

SEQ ID NO: 161 provides the nucleic acid sequence of primer no. 1005.

SEQ ID NO: 162 provides the amino acid sequence of Fab 0100 HC.

SEQ ID NO: 163 provides the amino acid sequence of Fab 0100 LC.

DESCRIPTION OF THE INVENTION

The invention relates to fusion proteins: proteins expressed from two ormore genes that have been joined artificially, for example viarecombinant technology or chemical coupling, and which originallyencoded separate proteins. Fusion proteins of the invention are capableof binding to a receptor that is only (in the sense non-ubiquitous)present on a platelet undergoing the conformational and functionalchanges associated with activation. Examples of such receptors mightoriginate from the alpha- or dense granules of resting platelets. Oneparticular example of such a receptor is TREM-like transcript 1 (TLT-1).

Triggering receptors expressed on myeloid cells (TREMs) have awell-established role in the biology of various myeloid lineages,playing important roles in the regulation of innate and adaptiveimmunity. TLT-1 belongs to the same family of proteins, though the TLT-1gene is expressed only in a single lineage, namely megakaryocytes andthrombocytes (platelets) and is exclusively found in the alpha-granulesof megakaryocytes and platelets. TLT-1 is a transmembrane protein thatis exposed on the surface of activated platelets upon alpha-granulerelease. To date, TLT-1 has not been found on the surface of restingplatelets or on the surface of any other cell types.

The extracellular portion of TLT-1 is known to consist of a single,immunoglobulin-like (Ig-like) domain connected to the platelet cellmembrane by a linker region called the stalk (Gattis et al., Jour BiolChem, 2006, Vol. 281, No. 19, pp. 13396-13403). The Ig-like domain ofhuman TLT-1 (hTLT-1) consists of 105 residues and is attached to themembrane by a 37-amino acid stalk. Thus, the Ig-like domain of TLT-1 isexpected to have considerable freedom of movement.

The putative transmembrane segment of hTLT-1 is 20 amino acids long.TLT-1 also has a cytoplasmic Immune-receptor Tyrosine-basedInhibitory-Motif, which may function as an intracellular signaltransduction domain.

The role of TLT-1 in platelet biology has not yet been fully elucidated;it is believed that TLT-1 plays a role in regulating coagulation andinflammation at the site of an injury. A soluble form of TLT-1containing the Ig-like domain has been reported (Gattis et al., JourBiol Chem, 2006, Vol. 281, No. 19, pp. 13396-13403). The specificfunctions of soluble versus platelet-bound TLT-1 remain to beestablished.

A receptor such as TLT-1 comprises epitopes that are useful targets forthe fusion proteins/constructs of the current invention. Fusion proteinsmay bind any part of TLT-1 that would be available for binding in vivo,such as surface accessible residues of the Ig-like domain, or part ofthe stalk. Hence, fusion proteins may bind one or more residues withinTLT-1 (20-125), TLT-1 (16-162), TLT-1 (126-162) and/or TLT-1 (129-142).

In a preferred embodiment, fusion proteins bind the stalk of TLT-1, suchas one or more residues of TLT-1 (126-162) or TLT-1 (129-142). Fusionproteins that bind to the stalk of TLT-1 are unlikely to interfere withthe function of the Ig-like domain and will probably not separate fromthe platelet surface if the Ig-like domain is shed. Furthermore, fusionproteins that bind the stalk of TLT-1 place their TF portion in afavorable position and orientation on the cell surface of activatedplatelets, relative to that of FVII and FVIIa. In another preferredembodiment, fusing TF to the C-terminal of an antibody, or fragmentthereof, will position TF even more favourably on the cell surface ofactivated platelets, relative to that of FVII and FVIIa.

In terms of the current invention, TLT-1 may be from any vertebrate,such as any mammal, such as a rodent (such as a mouse, rat or guineapig), a lagomorph (such as a rabbit), an artiodactyl (such as a pig,cow, sheep or camel) or a primate (such as a monkey or human being).TLT-1 is, preferably, human TLT-1. TLT-1 may be translated from anynaturally occurring genotype or allele that gives rise to a functionalTLT-1 protein. A non-limiting example of one human TLT-1 is thepolypeptide sequence of SEQ ID NO. 2.

Fusion proteins of the invention comprise a tissue factor-likecomponent. Tissue Factor is a 263 amino acid long, integral membraneglycoprotein receptor. It consists of an extracellular part folded intotwo compact fibronectin type III-like domains (1-209) that are eachstabilized by a single disulfide bond, a short linker (210-219), atransmembrane segment (220-242), and a short cytoplasmic tail (243-263).It forms a tight Ca²⁺-dependent complex with Factor VII/FVIIa.

In terms of the current invention, “tissue factor, or any biologicallyfunctional variant or fragment thereof”, may be any tissue factor-likepolypeptide that is able to bind Factor VII/VIIa, such that bloodcoagulation is stimulated. “Tissue factor” may be derived from anyvertebrate animal, such as any mammal, such as a rodent (such as amouse, rat or guinea pig), a lagomorph (such as a rabbit), anartiodactyl (such as a pig, cow, sheep or camel) or a primate (such as amonkey or a human being). “Tissue factor, or any biologically functionalvariant or fragment thereof” may be the extracellular domain of humantissue factor. “Tissue factor, or any biologically functional variant orfragment thereof” may be any polypeptide that is at least 90%, such asat least 91%, such as at least 92%, such as at least 93%, such as atleast 94%, such as at least 95%, such as at least 96%, such as at least97%, such as at least 98%, such as at least 99% identical to thepolypeptide sequence of tissue factor. “Tissue factor, or anybiologically functional variant or fragment thereof” may be anypolypeptide that is at least 90%, such as at least 91%, such as at least92%, such as at least 93%, such as at least 94%, such as at least 95%,such as at least 96%, such as at least 97%, such as at least 98%, suchas at least 99% identical to the polypeptide sequence of theextracellular domain of tissue factor or a variant thereof. “Tissuefactor, or any biologically functional variant or fragment thereof” maybe any polypeptide that is able to function as co-factor for FVII andFVIIa. Hence, “tissue factor or any biologically functional variant orfragment thereof” may be any polypeptide that is able to stimulate theamidolytic activity of FVIIa. Said “tissue factor, or any biologicallyfunctional variant or fragment thereof” may be the extracellular domainof TF (1-219). “Tissue Factor polypeptide” may be a polypeptidecomprising the soluble extracellular domain of Tissue Factor, i.e. aminoacids 1-219 (in the following referred to as sTF or sTF(1-219)), or afunctional variant or truncated form thereof. Preferably, the TissueFactor polypeptide at least comprises a fragment corresponding to theamino acid sequence 6-209 of Tissue Factor. Examples hereof aresTF(6-209), sTF(1-209), sTF (1-210), sTF (1-211), sTF (1-212), sTF(1-213), sTF (1-214), sTF (1-215), sTF (1-216), sTF (1-217), sTF(1-218), sTF(1-219), sTF(2-219), sTF(3-219), sTF(4-219), sTF(5-219).

In accordance with the current invention, “tissue factor, or anybiologically functional variant or fragment thereof” may have any one ormore of the features listed above.

Fusion proteins of the invention also comprise a “ligand”. The term“ligand” refers to any substance that is able to bind to and form acomplex with a biomolecule, in order to serve a biological purpose. Inone sense of the term, it is a signal triggering molecule binding to asite on a target protein by means of intermolecular forces such as ionicbonds, hydrogen bonds and Van der Waals forces. The association of aligand with said biomolecule is usually reversible. Binding of anaturally occurring ligand to its counterpart receptor may or may notalter the conformation of the receptor protein. In terms of the currentinvention, one object of said ligand is to target the TF-like componentto the surface of a platelet that is activated or in the process ofbeing activated.

The ligand may be any naturally occurring or synthetic ligand that bindsa receptor that is, preferably, only present on platelets undergoingactivation. The ligand of the current invention may be any naturallyoccurring or synthetic ligand that binds TLT-1, or the ligand may be anantibody, or fragment thereof, that has been raised against TLT-1. Theligand of the current invention may or may not result in a change in theconformational structure of TLT-1. Furthermore, the ligand of thecurrent invention may or may not result in intracellular signalling, asa result of binding to TLT-1. In a preferred embodiment, the ligand ofthe invention is capable of binding to the stalk of TLT-1. Hence, theligand of the current invention utilises a naturally occurring receptor,or portion thereof, in order to achieve the effect that is unique to andprovided by the current invention.

As mentioned above, the ligand component of the invented fusion proteinsmay be an antibody or a fragment thereof. The term “antibody” asreferred to herein includes whole antibodies and any antigen bindingfragment (i.e., “antigen-binding portion”) or single chains thereof. Anantibody refers to a glycoprotein comprising at least two heavy (H)chains and two light (L) chains inter-connected by disulfide bonds, oran antigen binding portion thereof. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as VH) and a heavy chainconstant region. Each light chain is comprised of a light chain variableregion (abbreviated herein as VL) and a light chain constant region. Thevariable regions of the heavy and light chains contain a binding domainthat interacts with an antigen. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). The constant regions of theantibodies may mediate the binding of the immunoglobulin to host tissuesor factors, including various cells of the immune system (e.g., effectorcells) and the first component (Clq) of the classical complement system.

An antibody of the invention may be a monoclonal antibody or apolyclonal antibody. In one embodiment, an antibody of the invention isa monoclonal antibody. An antibody of the invention may be a chimericantibody, a CDR-grafted antibody, a human or humanised antibody or anantigen binding portion of any thereof. For the production of bothmonoclonal and polyclonal antibodies, the experimental animal is asuitable mammal such as a goat, rabbit, rat or mouse.

A monoclonal antibody is, in structural terms, represented by a singlemolecular species having a single binding specificity and affinity for aparticular epitope. Monoclonal antibodies (mAbs) of the presentinvention can be produced by a variety of well-known techniques,including conventional monoclonal antibody methodology e.g., thestandard somatic cell hybridization technique of Kohler and Milstein(1975) Nature 256: 495, or viral or oncogenic transformation of Blymphocytes. The preferred animal system for preparing hybridomas is themurine system. Hybridoma production in the mouse is a verywell-established procedure. Immunization protocols and techniques forisolation of immunized splenocytes for fusion are known in the art.Fusion partners (e.g., murine myeloma cells) and fusion procedures arealso known.

To generate hybridomas producing monoclonal antibodies of the invention,splenocytes and/or lymph node cells from immunized mice can be isolatedand fused to an appropriate immortalized cell line, such as a mousemyeloma cell line. The resulting hybridomas can be screened for theproduction of antigen-specific antibodies. The antibody secretinghybridomas can be replated, screened again, and if still positive forsuitable IgG, the monoclonal antibodies can be subcloned at least twiceby limiting dilution. The stable subclones can then be cultured in vitroto generate small amounts of antibody in tissue culture medium forcharacterization.

An antibody of the invention may be prepared, expressed, created orisolated by recombinant means, such as (a) antibodies isolated from ananimal (e.g., a mouse) that is transgenic or transchromosomal for theimmunoglobulin genes of interest or a hybridoma prepared therefrom, (b)antibodies isolated from a host cell transformed to express the antibodyof interest, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of immunoglobulin gene sequences to other DNA sequences.

Suitable monoclonal antibodies, shown in table 1, are herein identifiedby means of the prefix “mAb” together with a 4-digit number. Hence, themonoclonal antibody may be mAb 0012 or a variant thereof. (Note that thevariable domain of the mAb referred to as “2F105” is identical to thatof mAb 0012.) The monoclonal antibody may be mAb 0023 or a variantthereof. The monoclonal antibody may be mAb 0051 or a variant thereof.The monoclonal antibody may be mAb 0061 or a variant thereof. Themonoclonal antibody may be mAb 0062 or a variant thereof. The monoclonalantibody may be mAb 0082 or a variant thereof.

TABLE 1 Non-limiting examples of suitable monoclonal antibodies mAb IDHC LC 0012 SEQ ID NO: 39 SEQ ID NO: 40 0023 SEQ ID NO: 41 SEQ ID NO: 420051 SEQ ID NO: 43 SEQ ID NO: 44 0052 SEQ ID NO: 45 SEQ ID NO: 46 0061SEQ ID NO: 47 SEQ ID NO: 48 0062 SEQ ID NO: 49 SEQ ID NO: 46 0082 SEQ IDNO: 50 SEQ ID NO: 48

The term “antigen-binding portion” of an antibody refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen, such as TLT-1 or another target receptor as describedherein. It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include a Fab fragment, aF(ab′)₂ fragment, a Fab′ fragment, a Fd fragment, a Fv fragment, a ScFvfragment, a dAb fragment and an isolated complementarity determiningregion (CDR). Single chain antibodies such as scFv and heavy chainantibodies such as VHH and camel antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments may be obtained using conventional techniquesknown to those of skill in the art, and the fragments may be screenedfor utility in the same manner as intact antibodies.

Suitable Fab fragments, shown in table 2, are herein identified by meansof the prefix “Fab” together with a 4-digit number. Said Fab fragmentmay be Fab 0012 or a variant thereof. Said Fab fragment may be Fab 0023or a variant thereof. Said Fab fragment may be Fab 0051 or a variantthereof. Said Fab fragment may be 0052 or a variant thereof. Said Fabfragment may be Fab 0061 or a variant thereof. Said Fab fragment may beFab 0062 or a variant thereof. Said Fab fragment may be Fab 0082 or avariant thereof.

TABLE 2 Non-limiting examples of suitable Fab fragments Fab ID VH-CH1 LC0012 SEQ ID NO: 51 SEQ ID NO: 40 0023 SEQ ID NO: 52 SEQ ID NO: 42 0051SEQ ID NO: 53 SEQ ID NO: 44 0052 SEQ ID NO: 54 SEQ ID NO: 46 0061 SEQ IDNO: 156 SEQ ID NO: 48 0082 SEQ ID NO: 55 SEQ ID NO: 48 AP-3 SEQ ID NO:56 SEQ ID NO: 57 AP-3.LC.C34S SEQ ID NO: 56 SEQ ID NO: 58

An antibody of the invention may be a human antibody or a humanisedantibody. The term “human antibody”, as used herein, is intended toinclude antibodies having variable regions in which both the frameworkand CDR regions are derived from human germline immunoglobulinsequences. Furthermore, if the antibody contains a constant region, theconstant region also is derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo). However, the term “human antibody”, asused herein, is not intended to include antibodies in which CDRsequences derived from the germline of another mammalian species, suchas a mouse, have been grafted onto human framework sequences.

Such a human antibody may be a human monoclonal antibody. Such a humanmonoclonal antibody may be produced by a hybridoma which includes a Bcell obtained from a transgenic nonhuman animal, e.g., a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene fused to an immortalized cell.

Human antibodies may be prepared by in vitro immunisation of humanlymphocytes followed by transformation of the lymphocytes withEpstein-Barr virus.

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody.

The term “humanized antibody” is intended to refer to antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. Additional framework region modifications may be made withinthe human framework sequences.

Antibodies of the invention can be tested for binding to the targetprotein by, for example, standard ELISA or Western blotting. An ELISAassay can also be used to screen for hybridomas that show positivereactivity with the target protein. The binding specificity of anantibody may also be determined by monitoring binding of the antibody tocells expressing the target protein, for example, by flow cytometry.

The specificity of an antibody of the invention for the target proteinmay be further studied by determining whether or not the antibody bindsto other proteins. For example, where it is desired to produce anantibody that specifically binds TLT-1 or a particular part, e.g.epitope, of TLT-1, the specificity of the antibody may be assessed bydetermining whether or not the antibody also binds to other molecules ormodified forms of TLT-1 that lack the part of interest.

Polypeptide or antibody “fragments” according to the invention may bemade by truncation, e.g. by removal of one or more amino acids from theN and/or C-terminal ends of a polypeptide. Up to 10, up to 20, up to 30,up to 40 or more amino acids may be removed from the N and/or C terminalin this way. Fragments may also be generated by one or more internaldeletions.

An antibody of the invention may be, or may comprise, a fragment of theanti-TLT-1 antibody or a variant thereof. The antibody of the inventionmay be or may comprise an antigen binding portion of this antibody or avariant thereof, as discussed further above. For example, the antibodyof the invention may be a Fab fragment of this antibody, or a variantthereof, or may be a single chain antibody derived from this antibody,or a variant thereof.

Antibodies, as well as fusion proteins that comprise an antibody, orfragment thereof, may be defined in terms of their epitopes and/orparatopes. The term “epitope” includes any protein determinant capableof specific binding to an immunoglobulin (or T-cell receptor). Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. An epitope having antigenic activity isa portion of a polypeptide to which an antibody immunospecificallybinds, as determined by any method well known in the art, for example,by immunoassays. Antigenic epitopes need not necessarily be immunogenic.

In terms of the current invention, “epitope” refers to the area orregion on an antigen (Ag), which is a receptor on an activated platelet,to which the antibody (Ab) portion of the fusion protein is capable ofspecifically binding, i.e. the area or region that is in physicalcontact with the Ab. An antigen's epitope may comprise amino acidresidues in the Ag that are directly involved in binding to a Ab (theimmunodominant component of the epitope) and other amino acid residues,which are not directly involved in the binding, such as amino acidresidues of the Ag which are effectively blocked by the Ab (in otherwords, the amino acid residue is within the “solvent-excluded surface”and/or the “footprint” of the Ab). The term epitope herein includes bothtypes of binding sites in any particular region of a receptor such asTLT-1 that specifically binds to an anti-TLT-1 antibody, or anotherTLT-1-specific agent according to the invention, unless otherwise stated(e.g., in some contexts the invention relates to antibodies that binddirectly to particular amino acid residues). Receptors such as TLT-1 maycomprise a number of different epitopes, which may include, withoutlimitation, (1) linear peptide antigenic determinants, (2)conformational antigenic determinants which consist of one or morenon-contiguous amino acids located near each other in the maturereceptor conformation; and (3) post-translational antigenic determinantswhich consist, either in whole or part, of molecular structurescovalently attached to TLT-1, such as carbohydrate groups.

The epitope for a given antibody (Ab)/antigen (Ag) pair can be definedand characterized at different levels of detail using a variety ofexperimental and computational epitope mapping methods. The experimentalmethods include mutagenesis, X-ray crystallography, Nuclear MagneticResonance (NMR) spectroscopy, Hydrogen deuterium eXchange MassSpectrometry (HX-MS) and various competition binding methods. As eachmethod relies on a unique principle, the description of an epitope isintimately linked to the method by which it has been determined. Thus,the epitope for a given Ab/Ag pair will be defined differently dependingon the epitope mapping method employed.

At its most detailed level, the epitope for the interaction between theAg and the Ab can be defined by the spatial coordinates defining theatomic contacts present in the Ag-Ab interaction, as well as informationabout their relative contributions to the binding thermodynamics. At aless detailed level the epitope can be characterized by the spatialcoordinates defining the atomic contacts between the Ag and Ab. At afurther less detailed level the epitope can be characterized by theamino acid residues that it comprises as defined by a specificcriterium, e.g. distance between atoms in the Ab and the Ag. At afurther less detailed level the epitope can be characterized throughfunction, e.g. by competition binding with other Abs. The epitope canalso be defined more generically as comprising amino acid residues forwhich substitution by another amino acid will alter the characteristicsof the interaction between the Ab and Ag.

In the context of an X-ray derived crystal structure defined by spatialcoordinates of a complex between an Ab, e.g. a Fab fragment, and its Ag,the term epitope is herein, unless otherwise specified or contradictedby context, specifically defined as platelet receptor residuescharacterized by having a heavy atom (i.e. a non-hydrogen atom) within adistance of 4 Å from a heavy atom in the Ab.

From the fact that descriptions and definitions of epitopes, dependenton the epitope mapping method used, are obtained at different levels ofdetail, it follows that comparison of epitopes for different Abs on thesame Ag can similarly be conducted at different levels of detail.

Epitopes described on the amino acid level, e.g. determined from anX-ray structure, are said to be identical if they contain the same setof amino acid residues. Epitopes are said to overlap if at least oneamino acid is shared by the epitopes. Epitopes are said to be separate(unique) if no amino acid residue is shared by the epitopes.

Epitopes characterized by competition binding are said to be overlappingif the binding of the corresponding Ab's are mutually exclusive, i.e. ifbinding of one Ab excludes simultaneous binding of the other Ab. Theepitopes are said to be separate (unique) if the Ag is able toaccommodate binding of both corresponding Ab's simultaneously. Thus,fusion proteins of the invention may be capable of binding to the sameepitope as mAb 0012. Fusion proteins may be capable of binding to thesame epitope as mAb 0023. Fusion proteins may be capable of binding tothe same epitope as mAb 0051. Fusion proteins may be capable of bindingto the same epitope as mAb 0061. Fusion proteins may be capable ofbinding to the same epitope as mAb 0062.

The epitope may comprise one or more residues selected from the groupconsisting of K133, I134, G135, S136, L137, A138, N140, A141, F142,5143, D144, P145 and A146 of SEQ ID NO: 4.

The epitope may comprise one or more residues selected from the groupconsisting of V17, Q18, C19, H20, Y21, R22, L23, Q24, D25, V26, K27,A28, L63, G64, G65, G66, L67, L68, G89, A90, R91, G92, P93, Q94, I95 andL96 of SEQ ID NO: 5.

The epitope may comprise one or more residues selected from the groupconsisting of L36, P37, E38, G39, C40, Q41, P42, L43, V44, S45, S46,A47, V73, T74, L75, Q76, E77, E78, D79, A80, G81, E82, Y83, G84, C85,M86, R91, G92, P93, Q94, I95, L96, H97, R98, V99, S100 and L101 of SEQID NO: 5.

The epitope may comprise one or more residues selected from the groupconsisting of V17, Q18, C19, H20, Y21, R22, L23, Q24, D25, V26, K27,A28, R91, G92, P93, Q94, I95, L96, H97, R98, V99, S100 and L101 of SEQID NO: 5.

The epitope may comprise one or more residues selected from the groupconsisting of E5, T6, H7, K8, I9, G10, S11, L12, A13, E14, N15, A16,F17, S18, D19, P20 and A21 of SEQ ID NO: 7.

The epitope may comprise one or more residues selected from the groupconsisting of K133, I134, G135, S136, L137, A138, N140, A141, F142,S143, D144, P145 and A146 of SEQ ID NO: 7.

The definition of the term “paratope” is derived from the abovedefinition of “epitope” by reversing the perspective. Thus, the term“paratope” refers to the area or region on the Ab to which an Agspecifically binds, i.e. to which it makes physical contact to the Ag.

The paratope may comprise one or more residues selected from the groupconsisting of H50, N52, Y56, H58, Y73, F79, S115, T116, V118 and Y120 ofthe anti-TLT-1 light (L) chain (SEQ ID NO: 40), and residues V20, F45,R49, Y50, W51, E68, T75, N77, S116, G117, V118 and T120 of theanti-TLT-1 heavy (H) chain (SEQ ID NO: 39)

In the context of an X-ray derived crystal structure defined by spatialcoordinates of a complex between an Ab, e.g. a Fab fragment, and its Ag,the term paratope is herein, unless otherwise specified or contradictedby context, specifically defined as Ag residues characterized by havinga heavy atom (i.e. a non-hydrogen atom) within a distance of 4 Å from aheavy atom in the platelet receptor.

The epitope and paratope for a given antibody (Ab)/antigen (Ag) pair maybe identified by routine methods. For example, the general location ofan epitope may be determined by assessing the ability of an antibody tobind to different fragments or variants of TLT-1. The specific aminoacids within TLT-1 that make contact with an antibody (epitope) and thespecific amino acids in an antibody that make contact with TLT-1(paratope) may also be determined using routine methods, such as thosedescribed in the examples. For example, the antibody and target moleculemay be combined and the Ab/Ag complex may be crystallised. The crystalstructure of the complex may be determined and used to identify specificsites of interaction between the antibody and its target.

Fusion proteins comprising a ligand that is an antibody or fragmentthereof may also be defined in terms of theircomplementarity-determining regions (CDRs). The term“complementarity-determining region” or “hypervariable region” when usedherein refers to the amino acid residues of an antibody that areresponsible for antigen binding. The complementarity-determining regionsor “CDRs” are generally comprised of amino acid residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain;(Kabat et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and/or those residues from a “hypervariableloop” (residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in theheavy-chain variable domain; Chothia and Lesk, 3. Mol. Biol 1987;196:901-917). Typically, the numbering of amino acid residues in thisregion is performed by the method described in Kabat et al., supra.Phrases such as “Kabat position”, “Kabat residue”, and “according toKabat” herein refer to this numbering system for heavy chain variabledomains or light chain variable domains. Using the Kabat numberingsystem, the actual linear amino acid sequence of a peptide may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include amino acid insertions (residue 52a,52b and 52c according to Kabat) after residue 52 of CDR H2 and insertedresidues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat)after heavy chain FR residue 82. The Kabat numbering of residues may bedetermined for a given antibody by alignment at regions of homology ofthe sequence of the antibody with a “standard” Kabat numbered sequence.

The term “framework region” or “FR” residues refer to those VH or VLamino acid residues that are not within the CDRs, as defined herein.

In one embodiment, the heavy chain comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (DYFMY) of SEQ ID NO:        41, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 69 to 85 (YISNGGDSSSYPDTVKG) of        SEQ ID NO: 41, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 118 to 129 (NKNWDDYYDMDY) of SEQ        ID NO: 41, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

In another embodiment, the light chain comprises:

-   -   a CDR1 sequence of amino acids 44 to 60 (KSSQSLLNSRTRKNYLA) of        SEQ ID NO: 42, wherein one, two, three or four of these amino        acids may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 76 to 82 (WASTRES) of SEQ ID NO:        42, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 115 to 122 (KQSYNLLT) of SEQ ID        NO: 42, wherein one or two of these amino acids may be        substituted with a different amino acid.

In another embodiment, the heavy chain comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (DYSMH) of SEQ ID NO:        43, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 69 to 85 (VISTYYGDVRYNQKFKG) of        SEQ ID NO: 43, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 118 to 129 (APMITTGAWFAY) of SEQ        ID NO: 43, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

In another embodiment, the light chain comprises:

-   -   a CDR1 sequence of amino acids 44 to 54 (KASQSVSNDVA) of SEQ ID        NO: 44, wherein one, two or three of these amino acids may be        substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 70 to 76 (YASSRYT) of SEQ ID NO:        44, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 109 to 117 (QQDYSSPYT) of SEQ ID        NO: 44, wherein one or two of these amino acids may be        substituted with a different amino acid.

In another embodiment, the heavy chain comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (SHWIE) of SEQ ID NO:        49, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 69 to 85 (EILPGSGNTNYNEKFKG) of        SEQ ID NO: 49, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 118 to 130 (GYYGLNYDWYFDV) of SEQ        ID NO: 49, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

In another embodiment, the light chain comprises:

-   -   a CDR1 sequence of amino acids 44 to 54 (RASQDISNYLN) of SEQ ID        NO: 46, wherein one, two or three of these amino acids may be        substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 70 to 76 (YTSRLHS) of SEQ ID NO:        46, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 109 to 117 (QQDTKLPYT) of SEQ ID        NO: 46, wherein one or two of these amino acids may be        substituted with a different amino acid.

In another embodiment, the heavy chain comprises:

-   -   a CDR1 sequence of amino acids 49 to 53 (RYWMT) of SEQ ID NO:        47, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 68 to 84 (EINPDSSTINYNPSLKD) of        SEQ ID NO: 47, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 117 to 121 (GVFTS) of SEQ ID NO:        47, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

In another embodiment, the light chain comprises:

-   -   a CDR1 sequence of amino acids 43 to 58 (RSSQSLVHRNGNTYFH) of        SEQ ID NO: 48, wherein one, two, three or four of these amino        acids may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 74 to 80 (KVSNRFS) of SEQ ID NO:        48, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 113 to 121 (SQSTHVPYT) of SEQ ID        NO: 48, wherein one or two of these amino acids may be        substituted with a different amino acid.

In another embodiment, the heavy chain comprises:

-   -   a CDR1 sequence of amino acids 49 to 53 (RYWMT) of SEQ ID NO:        39, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 68 to 84 (EINPDSSTINYTPSLKD) of        SEQ ID NO: 39, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 117 to 121 (GVFTS) of SEQ ID NO:        39, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

In another embodiment, the light chain comprises:

-   -   a CDR1 sequence of amino acids 43 to 58 (RSSQSLVHRNGNTYFH) of        SEQ ID NO: 40, wherein one, two, three or four of these amino        acids may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 74 to 80 (KVSNRFS) of SEQ ID NO:        40, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 113 to 121 (SQSTHVPYT) of SEQ ID        NO: 40, wherein one or two of these amino acids may be        substituted with a different amino acid.

In yet another embodiment, the heavy chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (NYWLG) of SEQ ID NO:        56, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 69 to 85 (DIYPGGGYNKYNENFKG) of        SEQ ID NO: 56, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 118 to 128 (EYGNYDYAMDS) of SEQ        ID NO: 56, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

In a further embodiment, the light chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 44 to 59 (RSSRSLLHSNGNTYLC) of        SEQ ID NO: 57, wherein one, two, three or four of these amino        acids may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 75 to 81 (RMSNLAS) of SEQ ID NO:        57, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 114 to 122 (MQHLEYPFT) of SEQ ID        NO: 57, wherein one or two of these amino acids may be        substituted with a different amino acid.

Hence, the construct of the current invention is any fusion protein orchimer that comprises (i) at least one TF, or biologically functionalvariant(s) or fragment(s) thereof, and (ii) a ligand that is capable ofbinding (iii) a receptor, and/or a fragment thereof, wherein thereceptor is present only (in the sense of non-ubiquitous) on the surfaceof activated platelets. In one preferred embodiment, (iii) is TLT-1. Thefusion protein (construct) of the current invention is preferablyengineered such that its constituent parts may function independently ofone another. For example, said “tissue factor . . . ” component of thecurrent invention is able to bind FVII and FVIIa, as opposed to beingsterically hindered from doing so due to the presence of said “ligand”component of the invention. Likewise, said “ligand” component of theinvention is preferably able to bind a receptor such as TLT-1,unhindered by the presence of said “tissue factor . . . ” component. Thecarboxy terminus of the “TF polypeptide” component may be covalentlyattached to the amino terminus of the ligand component of the construct,or vice versa. Said ligand component of the construct will preferablynot bind any other TREM. The construct of the current invention may ormay not comprise a linker between said TF and said ligand constituents.Said optional linker may be any one of the linkers described in Table 3or may be any other linker that binds both TF and ligand constituentparts of the construct, such that both are functional.

The fusion protein/construct of the present invention may comprise (i)tissue factor and (ii) any ligand that is capable of binding (iii)TLT-1. Said fusion protein/construct may further comprise a linker,which may be any one of linkers L1-L10 provided in Table 3.

The construct of the present invention may comprise (i) tissue factorand (ii) an antibody capable of binding (iii) TLT-1. Said antibody maybe a monoclonal antibody. The construct may further comprise a linker.Said linker may be any one of linkers L1-L10 provided in Table 3.

The construct of the present invention may comprise (i) tissue factorand (ii) a Fab fragment capable of binding (iii) TLT-1.

The construct may further comprise a linker, which may be any one oflinkers (L1-L10) provided in Table 3.

The construct of the present invention may comprise (i) tissue factorand (ii) a F(ab′)₂ fragment capable of binding (iii) TLT-1. Theconstruct may further comprise a linker, which may be any one of thelinkers (L1-L10) provided in Table 3.

The construct of the present invention may comprise (i) tissue factorand (ii) a Fab′ fragment capable of binding (iii) TLT-1. Said constructmay further comprise a linker, which may be any one of the linkers(L1-L10) provided in Table 3.

The construct of the present invention may comprise (i) tissue factorand (ii) a Fd fragment capable of binding (iii) TLT-1. Said constructmay further comprise a linker, which may be any one of the linkers(L1-L10) provided in Table 3.

The construct of the present invention may comprise (i) tissue factorand (ii) a Fv fragment capable of binding (iii) TLT-1. Said constructmay further comprise a linker, which may be any one of linkers (L1-L10)provided in Table 3.

The construct of the present invention may comprise (i) tissue factorand (ii) a dAb fragment capable of binding (iii) TLT-1. Said constructmay further comprise a linker, which may be any one of linkers (L1-L10)provided in Table 3.

The construct of the present invention may comprise (i) tissue factorand (ii) an isolated complementarity determining region (CDR) capable ofbinding (iii) TLT-1. Said construct may further comprise a linker, whichmay be any one of the linkers (L1-L10) provided in Table 3.

The construct of the present invention may comprise (i) any biologicallyfunctional variant or fragment of tissue factor and (ii) any ligandcapable of binding (iii) TLT-1. Said construct may further comprise alinker, which may be any one of linkers (L1-L10) provided in Table 3.

The construct of the present invention may comprise (i) any biologicallyfunctional variant or fragment of tissue factor and (ii) an antibodycapable of binding (iii) TLT-1. Said construct may further comprise alinker, which may be any one of linkers (L1-L10) provided in Table 3.

The construct of the present invention may comprise (i) any biologicallyfunctional variant or fragment of tissue factor and (ii) a Fab fragmentcapable of binding (iii) TLT-1. Said construct may further comprise alinker, which may be any one of linkers (L1-L10) provided in Table 3.

The construct of the present invention may comprise (i) any biologicallyfunctional variant or fragment of tissue factor and (ii) a F(ab′)₂fragment capable of binding (iii) TLT-1. Said construct may furthercomprise a linker, which may be any one of the linkers (L1-L10) providedin Table 3.

The construct of the present invention may comprise (i) any biologicallyfunctional variant or fragment of tissue factor and (ii) a Fab′ fragmentcapable of binding (iii) TLT-1. Said construct may further comprise alinker, which may be any one of the linkers (L1-L10) provided in Table3.

The construct of the present invention may comprise (i) any biologicallyfunctional variant or fragment of tissue factor and (ii) a Fd fragmentcapable of binding (iii) TLT-1. Said construct may further comprise alinker, which may be any one of the linkers (L1-L10) provided in Table3.

The construct of the present invention may comprise (i) any biologicallyfunctional variant or fragment of tissue factor and (ii) a Fv fragmentcapable of binding (iii) TLT-1. Said construct may further comprise alinker, which may be any one of the linkers (L1-L10) provided in Table3.

The construct of the present invention may comprise (i) any biologicallyfunctional variant or fragment of tissue factor and (ii) a dAb fragmentcapable of binding (iii) TLT-1. Said construct may further comprise alinker, which may be any one of the linkers (L1-L10) provided in Table3.

The construct of the present invention may comprise (i) any biologicallyfunctional variant or fragment of tissue factor and (ii) an isolatedcomplementarity determining region (CDR) capable of binding (iii) TLT-1.Said construct may further comprise a linker, which may be any one ofthe linkers (L1-L10) provided in Table 3.

The construct of the present invention may comprise (i) theextracellular domain of tissue factor and (ii) any ligand capable ofbinding (iii) TLT-1. Said construct may further comprise a linker. Saidlinker may be any one of linkers L1-L10 provided in Table 3.

The construct of the present invention may comprise (i) theextracellular domain of tissue factor and (ii) an antibody capable ofbinding (iii) TLT-1. Said construct may further comprise a linker. Saidlinker may be any one of linkers L1-L10 provided in Table 3.

The construct of the present invention may comprise (i) theextracellular domain of tissue factor and (ii) a Fab fragment capable ofbinding (iii) TLT-1. Said construct may further comprise a linker. Saidlinker may be any one of linkers L1-L10 provided in Table 3.

The construct of the present invention may comprise (i) theextracellular domain of tissue factor and (ii) a F(ab′)₂ fragmentcapable of binding (iii) TLT-1. Said construct may further comprise alinker. Said linker may be any one of the linkers (L1-L10) provided inTable 3.

The construct of the present invention may comprise (i) theextracellular domain of tissue factor and (ii) a Fab′ fragment capableof binding (iii) TLT-1. Said construct may further comprise a linker.Said linker may be any one of the linkers (L1-L10) provided in Table 3.

The construct of the present invention may comprise (i) theextracellular domain of tissue factor and (ii) a Fd fragment capable ofbinding (iii) TLT-1. Said construct may further comprise a linker. Saidlinker may be any one of the linkers (L1-L10) provided in Table 3.

The construct of the present invention may comprise (i) theextracellular domain of tissue factor and (ii) a Fv fragment capable ofbinding (iii) TLT-1. Said construct may further comprise a linker. Saidlinker may be any one of the linkers (L1-L10) provided in Table 3.

The construct of the present invention may comprise (i) theextracellular domain of tissue factor and (ii) a dAb fragment capable ofbinding (iii) TLT-1. Said construct may further comprise a linker. Saidlinker may be any one of the linkers (L1-L10) provided in Table 3.

The construct of the present invention may comprise (i) theextracellular domain of tissue factor and (ii) an isolatedcomplementarity determining region (CDR) capable of binding (iii) TLT-1.Said construct may further comprise a linker. Said linker may be any oneof the linkers (L1-L10) provided in Table 3.

The construct of the current invention may be a fusion proteincomprising the variable domain of mAb 0012 (2F105) HC, the human IgG4CH1 constant region, the glycine-serine linker and the extracellulardomain of human Tissue Factor (2F105HC-V-CH1-linker-hTF ECD).

The construct of the current invention may be a fusion proteinconsisting of the variable domain of mAb 0012 (2F105) HC, the human IgG4CH1 constant region, the glycine-serine linker and the extracellulardomain of human Tissue Factor (2F105HC-V-CH1-linker-hTF ECD).

As mentioned above, the fusion protein of the current invention maycomprise a linker. Non-limiting examples of linker amino acid sequencesare shown in Table 3. Hence, said linker may be L1. The linker may beL2. The linker may be L3. The linker may be L4. The linker may be L5.The linker may be L6. The linker may be L7. The linker may be L8. Thelinker may be L9. The linker may be L10.

TABLE 3 Non-limiting examples of optional linkers Linker ID Length (AA)Linker sequence L0  0 no linker L1  2 GS L2  7 GSGGGGS L3 12GSGGGGSGGGGS L4a 17 GSGGGGSGGGGSGGGGS L4b 17 GGGGSGGGGSGGGGSGS L5 22GGGGSGSGGGGSGGGGSGGGGS L6 27 GGGGSGGGGSGSGGGGSGGGGSGGGGS L7 32GGGGSGGGGSGGGGSGSGGGGSGGGGSGGGGS L8 37GGGGSGGGGSGGGGSGGGGSGSGGGGSGGGGSGGGGS L9 42GGGGSGGGGSGGGGSGGGGSGGGGSGSGGGGSGGGGSGGGGS L10 16 YGPPSPSSPAPEFLGG

As mentioned above, the extracellular part of TLT-1 consists of animmunoglobulin-like domain and a stalk. Fusion proteins of the inventionmay be capable of binding either of these. When part (ii) of the fusionprotein is capable of binding the immunoglobulin-like domain, a longerlinker may allow part (i) of said fusion protein to adapt a functionallyrelevant position and orientation on the surface of the activatedplatelet, thereby facilitating its function. This is because part (i),i.e. the TF polypeptide, must be in the special vicinity of and properlyoriented relative to FVII/FVIIa: TF acts as co-factor to FVII/FVIIa,which binds Ca²⁺ and phospholipid on the surface of activated platelets.

A fusion protein that is capable of binding the stalk of TLT-1 isadjacent to the platelet membrane. Hence, a fusion protein that iscapable of binding the stalk may comprise a linker; however, theinclusion of a linker does not necessarily affect the function of the TFpart of the fusion protein.

Examples of suitable fusion proteins, wherein (ii) is a monoclonalantibody, are provided in table 4. As each mAb has two identical heavychains (HC) and two identical light chains (LC), fusion proteins whereinpart (ii) is a mAb may comprise two TF polypeptides (part (i)). TF maybe fused to a HC of the mAb; TF may be fused to a LC of the mAb. TF maybe fused to a ligand which, in turn, is fused to a HC of the mAb or a LCof the mAb. Following are examples of how to interpret the names of thefusion proteins provided in table 4:

In fusion protein “mAb 0012-(HC-L0-hTF.1-219)₂;LC₂”:

-   -   “mAb 0012”: monoclonal antibody 0012.    -   “(HC-L0-hTF.1-219)₂”: one hTF.1-219 is fused to each HC; the        N-terminal of hTF.1-219 is fused to the C-terminal of the heavy        chain.    -   “L0”: no linker is present.    -   LC₂: there are two light chains, to which nothing is fused.        In fusion protein “mAb 0023-(HC-L4a-hTF. 1-219)₂;(LC-HPC4)₂”:    -   “mAb 0023” is monoclonal antibody 0023.    -   “(HC-L4a-hTF.1-219)₂” indicates that one hTF.1-219 is fused to        each HC via a linker; the N-terminal of hTF.1-219 is fused to        the C-terminal of linker L4a, whose N-terminal is fused to the        C-terminal of the heavy chain of the mAb.    -   “(LC-HPC4)₂” indicates that an HPC4 tag is fused to the        C-terminal of each LC.        In fusion protein “mAb 0012-(LC-L5-hTF.1-219)₂;HC₂”:    -   (LC-L5-hTF.1-219)₂ indicates that the N-terminal of hTF.1-219 is        fused to the C-terminal of linker L5 whose N-terminal is fused        to the C-terminal of the light chain.    -   HC₂ indicates that there are two heavy chains, to which nothing        is fused.        In fusion protein “mAb 0012-(hTF.1-219-L4b-LC)₂;HC₂”:    -   (hTF.1-219-L4b-LC)₂ indicates that the C-terminal of hTF.1-219        is fused to the linker L4b which is fused to the N-terminal end        of the light chain.

TABLE 4 Non-limiting examples of mAb-TF fusion proteins mAb-hTF.1-219fusion protein ID mAb-hTF.1-219 fusion protein name 0116 mAb0012-(HC-L0-hTF.1-219)₂; LC₂ 0086 mAb 0012-(HC-L1-hTF.1-219)₂; LC₂ 0087mAb 0012-(HC-L2-hTF.1-219)₂; LC₂ 0088 mAb 0012-(HC-L3-hTF.1-219)₂; LC₂0018 mAb 0012-(HC-L4a-hTF.1-219)₂; (LC-HPC4)₂ 0013 mAb0012-(hTF.1-219-L4b-HC)₂; LC₂ 0089 mAb 0012-(HC-L5-hTF.1-219)₂; LC₂ 0090mAb 0012-(HC-L6-hTF.1-219)₂; LC₂ 0091 mAb 0012-(HC-L7-hTF.1-219)₂; LC₂0092 mAb 0012-(HC-L8-hTF.1-219)₂; LC₂ 0093 mAb 0012-(HC-L9-hTF.1-219)₂;LC₂ 0107 mAb 0012-(LC-L0-hTF.1-219)₂; HC₂ 0108 mAb0012-(LC-L1-hTF.1-219)₂; HC₂ 0109 mAb 0012-(LC-L2-hTF.1-219)₂; HC₂ 0045mAb 0012-(LC-L3-hTF.1-219)₂; HC₂ 0019 mAb 0012-(LC-L4a-hTF.1-219)₂; HC₂0025 mAb 0012-(hTF.1-219-L4b-LC)₂; HC₂ 0046 mAb 0012-(LC-L5-hTF.1-219)₂;HC₂ 0047 mAb 0012-(LC-L6-hTF.1-219)₂; HC₂ 0048 mAb0012-(LC-L7-hTF.1-219)₂; HC₂ 0049 mAb 0012-(LC-L8-hTF.1-219)₂; HC₂ 0050mAb 0012-(LC-L9-hTF.1-219)₂; HC₂ 0034 mAb 0023-(HC-L4a-hTF.1-219)₂;(LC-HPC4)₂ 0035 mAb 0023-(LC-L4a-hTF.1-219)₂; HC₂ 0056 mAb0051-(HC-L4a-hTF.1-219)₂; (LC-HPC4)₂ 0055 mAb 0051-(LC-L4a-hTF.1-219)₂;HC₂ 0060 mAb 0052-(HC-L4a-hTF.1-219)₂; (LC-HPC4)₂ 0059 mAb0052-(LC-L4a-hTF.1-219)₂; HC₂

Examples of suitable fusion proteins wherein (ii) is a Fab fragment areprovided in table 5. TF may be fused to a HC of the mAb; TF may be fusedto a LC of the mAb. TF may be fused to a ligand which, in turn, is fusedto a HC of the mAb or a LC of the mAb. Following are examples of how tointerpret the names of the fusion proteins provided in table 5:

In fusion protein “Fab 0012-V_(H)-CH1-L0-hTF. 1-219;LC-HPC4”:

-   -   “Fab 0012”: Fab fragment of mAb 0012.    -   “V_(H)-CH1-L0-hTF.1-219”: the N-terminal of hTF.1-219 is        directly fused to the C-terminal of the V_(H)-CH1 domain of the        Fab fragment.    -   “HPC4”: a purification tag is at the N-terminal of the light        chain.

In fusion protein “Fab 0012-hTF.1-219-L4b-V_(H)-CH1;LC-HPC4”:

-   -   “hTF.1-219-L4b-V_(H)-CH1”: indicates that the C-terminal of        tissue factor is fused to the N-terminal of linker 4b which is        fused to the V_(H)-CH1 domain of the Fab fragment.

TABLE 5 Non-limiting examples of Fab-TF fusion proteins Fab-hTF.1-219fusion protein ID Fab-hTF.1-219 fusion protein name 0073 Fab0012-V_(H)-CH1-L0-hTF.1-219; LC-HPC4 0011 Fab0012-V_(H)-CH1-L4a-hTF.1-219; LC-HPC4 0014 Fab0012-hTF.1-219-L4b-V_(H)-CH1; LC-HPC4 0057 Fab0012-V_(H)-CH1-L10-hTF.1-219; LC-HPC4 0105 Fab0061-V_(H)-CH1-L10-hTF.1-219; LC-HPC4 0106 Fab0082-V_(H)-CH1-L10-hTF.1-219; LC-HPC4 0070 Fab 0012-LC-L0-hTF.1-219;V_(H)-CH1-HPC4 0071 Fab 0012-LC-L1-hTF.1-219; V_(H)-CH1-HPC4 0072 Fab0012-LC-L2-hTF.1-219; V_(H)-CH1-HPC4 0039 Fab 0012-LC-L3-hTF.1-219;V_(H)-CH1-HPC4 0020 Fab 0012-LC-L4a-hTF.1-219; V_(H)-CH1-HPC4 0024 Fab0012-hTF.1-219-L4b-LC; V_(H)-CH1-HPC4 0040 Fab 0012-LC-L5-hTF.1-219;V_(H)-CH1-HPC4 0041 Fab 0012-LC-L6-hTF.1-219; V_(H)-CH1-HPC4 0042 Fab0012-LC-L7-hTF.1-219; V_(H)-CH1-HPC4 0043 Fab 0012-LC-L8-hTF.1-219;V_(H)-CH1-HPC4 0044 Fab 0012-LC-L9-hTF.1-219; V_(H)-CH1-HPC4 0063 Fab0023-LC-L3-hTF.1-219; V_(H)-CH1-HPC4 0038 Fab 0023-LC-L4a-hTF.1-219;V_(H)-CH1-HPC4 0064 Fab 0023-LC-L5-hTF.1-219; V_(H)-CH1-HPC4 0065 Fab0023-LC-L6-hTF.1-219; V_(H)-CH1-HPC4 0066 Fab 0023-LC-L7-hTF.1-219;V_(H)-CH1-HPC4 0067 Fab 0023-LC-L8-hTF.1-219; V_(H)-CH1-HPC4 0068 Fab0023-LC-L9-hTF.1-219; V_(H)-CH1-HPC4 0033 Fab0023-V_(H)-CH1-L4a-hTF.1-219; LC-HPC4 0053 Fab 0051-LC-L4a-hTF.1-219;V_(H)-CH1-HPC4 0054 Fab 0051-V_(H)-CH1-L4a-hTF.1-219; LC-HPC4 0069 Fab0052-LC-L4a-hTF.1-219; V_(H)-CH1-HPC4 0058 Fab0052-V_(H)-CH1-L4a-hTF.1-219; LC-HPC4

As described above, fusion proteins of the invention are capable ofbinding a receptor that is present on platelets undergoing activation,such as TLT-1. The term “binding affinity” is intended to refer to theproperty of fusion proteins, or the antibody component of fusionproteins to bind or not to bind to their target. Binding affinity may bequantified by determining the binding constant (K_(D)) for an antibodycomponent and its target. Similarly, the specificity of binding of anantibody component to its target may be defined in terms of thecomparative binding constants (K_(D)) of the antibody for its target ascompared to the binding constant with respect to the antibody andanother, non-target molecule.

Typically, the K_(D) for the antibody with respect to the target will be2-fold, preferably 5-fold, more preferably 10-fold less than K_(D) withrespect to the other, non-target molecule such as unrelated material oraccompanying material in the environment. More preferably, the K_(D)will be 50-fold less, even more preferably 100-fold less, and yet morepreferably 200-fold less.

The value of this binding constant can be determined directly bywell-known methods, and can be computed even for complex mixtures bymethods such as those, for example, set forth in Caceci et al. (Byte9:340-362, 1984). For example, the K_(D) may be established using adouble-filter nitrocellulose filter binding assay such as that disclosedby Wong & Lohman (Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993). Otherstandard assays to evaluate the binding ability of ligands such asantibodies towards targets are known in the art, including for example,ELISAs, Western blots, RIAs, and flow cytometry analysis. The bindingkinetics (e.g., binding affinity) of the antibody can also be assessedby standard assays known in the art, such as by surface plasmon resonce(SPR) analysis.

A competitive binding assay can be conducted in which the binding of theantibody to the target is compared to the binding of the target byanother, known ligand of that target, such as another antibody.

K_(D) values for the ligand, such as an antibody or fragment thereof, ofthe invention may also be at least 1×10⁻¹⁵M, such as at least 1×10⁻¹⁴M,such as at least 1×10⁻¹³M, such as at least 1×10⁻¹²M, such as at least1×10⁻¹¹M, such as at least 1×10⁻¹⁰M, such as approximately 3×10⁻⁹M, suchas at least 1×10⁻⁹M, or at least 1×10⁻⁸M. An antibody of the inventionmay have a Kd (or Ki) for its target of 1×10⁻⁷M or less, 1×10⁻⁸M or lessor 1×10⁻⁹M or less.

Preferred K_(D) values for the ligand of the invention, such as anantibody or fragment thereof, may be 1×10⁻¹⁵M to 1×10⁻¹⁴M, such as1×10⁻¹⁴M to 1×10⁻¹³M 1×10⁻¹³M to 1×10⁻¹²M, such as 1×10⁻¹²M to 1×10⁻¹¹M,such as 1×10⁻¹¹M to 1×10⁻¹⁰M, such as 1×10⁻¹⁰M to 1×10⁻⁹M such asapproximately 3×10⁻⁹M, such as 1×10⁻⁹M to 2×10⁻⁸M.

An antibody that specifically binds its target may bind its target witha high affinity, such as a K_(D) as discussed above, and may bind toother, non-target molecules with a lower affinity. For example, theantibody may bind to a non-target molecules with a K_(D) of 1×10⁻⁶M ormore, more preferably 1×10⁻⁵ M or more, more preferably 1×10⁻⁴M or more,more preferably 1×10⁻³ M or more, even more preferably 1×10⁻² M or more.An antibody of the invention is preferably capable of binding to itstarget with an affinity that is at least two-fold, 10-fold, 50-fold,100-fold, 200-fold, 500-fold, 1,000-fold or 10,000-fold or greater thanits affinity for binding to another non-target molecule, such as otherTREMs than TLT-1.

As mentioned above, fusion proteins may comprise a tissue factor-likecomponent that is at least 90% identical to the extracellular domain oftissue factor or a variant thereof. The term “identity”, as known in theart, refers to a relationship between the sequences of two or morepolypeptides, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptides, as determined by the number of matches between strings oftwo or more amino acid residues. “Identity” measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by a particular mathematical model orcomputer program (i.e., “algorithms”). Identity of related polypeptidescan be readily calculated by known methods. Such methods include, butare not limited to, those described in Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987;Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M.Stockton Press, New York, 1991; and Carillo et al., SIAM J. AppliedMath. 48, 1073 (1988).

Preferred methods for determining identity are designed to give thelargest match between the sequences tested. Methods of determiningidentity are described in publicly available computer programs.Preferred computer program methods for determining identity between twosequences include the GCG program package, including GAP (Devereux etal., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group,University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA(Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX programis publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well knownSmith Waterman algorithm may also be used to determine identity.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3.times. the average diagonal; the “average diagonal” is the averageof the diagonal of the comparison matrix being used; the “diagonal” isthe score or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 1/10 times the gap opening penalty), as well as a comparisonmatrix such as PAM 250 or BLOSUM 62 are used in conjunction with thealgorithm. A standard comparison matrix (see Dayhoff et al., Atlas ofProtein Sequence and Structure, vol. 5, supp. 3 (1978) for the PAM 250comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci USA 89,10915-10919 (1992) for the BLOSUM 62 comparison matrix) is also used bythe algorithm.

Preferred parameters for a peptide sequence comparison include thefollowing: Algorithm: Needleman et al., 3. Mol. Biol. 48, 443-453(1970); Comparison matrix: BLOSUM 62 from Henikoff et al., PNAS USA 89,10915-10919 (1992); Gap Penalty: 12, Gap Length Penalty: 4, Threshold ofSimilarity: 0.

The GAP program is useful with the above parameters. The aforementionedparameters are the default parameters for peptide comparisons (alongwith no penalty for end gaps) using the GAP algorithm.

The functional effects of the invented fusion proteins may be assessedby means of various in vitro and in vivo experiments. In vitroexperiments may be designed to assess the function of the fusionproteins as a whole, as well as their component (i) TF and (ii) ligandparts. Such experiments are described in detail in the examples. Theseinclude methods of testing the ability of:

-   -   fusion proteins to bind FVII/FVIIa;    -   fusion proteins' TF component to selectively enhance        FVIIa-mediated FX activation on activated platelets;    -   fusion proteins' TF component to enhance FVIIa-mediated FX        activation on TLT-1-enriched phospholipid vesicles;    -   fusion proteins to promote fibrin clot formation in        hemophilia-like platelet-rich plasma    -   fusion proteins to promote fibrin clot formation in        hemophilia-like whole blood;        In vivo, fusion proteins may be tested in a tail-bleeding model        in haemophilic mice that are transfused with human platelets.        Furthermore in vivo, fusion proteins may be tested in a        tail-bleeding model in haemophilic mice with the human TLT-1        gene inserted (“humanized” with respect to TLT-1).

The invention also relates to polynucleotides that encode antibodies ofthe invention. Thus, a polynucleotide of the invention may encode anyantibody as described herein. The terms “nucleic acid molecule” and“polynucleotide” are used interchangeably herein and refer to apolymeric form of nucleotides of any length, either deoxyribonucleotidesor ribonucleotides, or analogs thereof. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, messenger RNA (mRNA),cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA ofany sequence, isolated RNA of any sequence, nucleic acid probes, andprimers. A polynucleotide of the invention may be provided in isolatedor purified form.

A nucleic acid sequence which “encodes” a selected polypeptide is anucleic acid molecule which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Forthe purposes of the invention, such nucleic acid sequences can include,but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,genomic sequences from viral or prokaryotic DNA or RNA, and evensynthetic DNA sequences. A transcription termination sequence may belocated 3′ to the coding sequence.

Polynucleotides of the invention can be synthesised according to methodswell known in the art, as described by way of example in Sambrook et al(1989, Molecular Cloning—a laboratory manual; Cold Spring Harbor Press).

The nucleic acid molecules of the present invention may be provided inthe form of an expression cassette which includes control sequencesoperably linked to the inserted sequence, thus allowing for expressionof the antibody of the invention in vivo. These expression cassettes, inturn, are typically provided within vectors (e.g., plasmids orrecombinant viral vectors). Such an expression cassette may beadministered directly to a host subject. Alternatively, a vectorcomprising a polynucleotide of the invention may be administered to ahost subject. Preferably the polynucleotide is prepared and/oradministered using a genetic vector. A suitable vector may be any vectorwhich is capable of carrying a sufficient amount of genetic information,and allowing expression of a polypeptide of the invention.

The present invention thus includes expression vectors that comprisesuch polynucleotide sequences. Such expression vectors are routinelyconstructed in the art of molecular biology and may for example involvethe use of plasmid DNA and appropriate initiators, promoters, enhancersand other elements, such as for example polyadenylation signals whichmay be necessary, and which are positioned in the correct orientation,in order to allow for expression of a peptide of the invention. Othersuitable vectors would be apparent to persons skilled in the art. By wayof further example in this regard we refer to Sambrook et al.

The invention also includes isolated cells that have been modified toexpress a construct according to the invention. Such cells includetransient, or—preferably—stable higher eukaryotic cell lines, such asmammalian cells or insect cells; lower eukaryotic cells, such as yeast;or prokaryotic cells such as bacterial cells. Particular examples ofcells which may be modified by insertion of vectors or expressioncassettes encoding for a construct of the invention include mammalianHEK293T, CHO, HeLa and COS cells. Preferably the cell line selected willbe one which is not only stable, but also allows for matureglycosylation and cell surface expression of a polypeptide.

Such cell lines of the invention may be cultured using routine methodsto produce a fusion protein, antibody or construct according to theinvention. Alternatively, polynucleotides, expression cassettes orvectors of the invention may be administered to a cell from a subject exvivo and the cell then returned to the body of the subject.

In another aspect, the present invention provides compositions andformulations comprising molecules of the invention, such as the fusionproteins, polynucleotides, vectors and cells described herein. Forexample, the invention provides a pharmaceutical composition thatcomprises one or more fusion proteins of the invention, formulatedtogether with a pharmaceutically acceptable carrier.

Accordingly, one object of the invention is to provide a pharmaceuticalformulation comprising such an antibody which is present in aconcentration from 0.25 mg/ml to 250 mg/ml, and wherein said formulationhas a pH from 2.0 to 10.0. The formulation may further comprise a buffersystem, preservative(s), tonicity agent(s), chelating agent(s),stabilizers and surfactants. The use of preservatives, isotonic agents,chelating agents, stabilizers and surfactants in pharmaceuticalcompositions is well-known to the skilled person. Reference may be madeto Remington: The Science and Practice of Pharmacy, 19^(th) edition,1995.

In one embodiment, the pharmaceutical formulation is an aqueousformulation. Such a formulation is typically a solution or a suspension.The terms “aqueous formulation” is defined as a formulation comprisingat least 50% w/w water. Likewise, the term “aqueous solution” is definedas a solution comprising at least 50% w/w water, and the term “aqueoussuspension” is defined as a suspension comprising at least 50% w/wwater.

In another embodiment, the pharmaceutical formulation is a freeze-driedformulation, to which the physician or the patient adds solvents and/ordiluents prior to use.

In a further aspect, the pharmaceutical formulation comprises an aqueoussolution of such an antibody, and a buffer, wherein the antibody ispresent in a concentration from 1 mg/ml or above, and wherein saidformulation has a pH from about 2.0 to about 10.0.

The term “treatment”, as used herein, refers to the medical therapy ofany human or other animal subject in need thereof. Said subject isexpected to have undergone physical examination by a [veterinary]medical practitioner, who has given a tentative or definitive diagnosiswhich would indicate that the use of said specific treatment isbeneficial to the health of said human or other animal subject. Thetiming and purpose of said treatment may vary from one individual toanother, according to the status quo of the subject's health. Thus, saidtreatment may be prophylactic, palliative, symptomatic and/or curative.

In terms of the present invention, prophylactic, palliative, symptomaticand/or curative treatments may represent separate aspects of theinvention.

A coagulopathy that results in an increased haemorrhagic tendency may becaused by any qualitative or quantitative deficiency of anypro-coagulative component of the normal coagulation cascade or anyupregulation of fibrinolysis. Such coagulopathies may be congenitaland/or acquired and/or iatrogenic and are identified by a person skilledin the art.

Non-limiting examples of congenital hypocoagulopathies are haemophiliaA, haemophilia B, Factor VII deficiency, Factor X deficiency, Factor XIdeficiency, von Willebrand's disease and thrombocytopenias such asGlanzmann's thombasthenia and Bernard-Soulier syndrome.

A non-limiting example of an acquired coagulopathy is serine proteasedeficiency caused by vitamin K deficiency; such vitamin K-deficiency maybe caused by administration of a vitamin K antagonist, such as warfarin.Acquired coagulopathy may also occur following extensive trauma. In thiscase otherwise known as the “bloody vicious cycle”, it is characterisedby haemodilution (dilutional thrombocytopaenia and dilution of clottingfactors), hypothermia, consumption of clotting factors and metabolicderangements (acidosis). Fluid therapy and increased fibrinolysis mayexaserbate this situation. Said haemorrhage may be from any part of thebody.

Haemophilia A with “inhibitors” (that is, allo-antibodies against factorVIII) and haemophilia B with “inhibitors” (that is, allo-antibodiesagainst factor IX) are non-limiting examples of coagulopathies that arepartly congenital and partly acquired.

A non-limiting example of an iatrogenic coagulopathy is an overdosage ofanticoagulant medication—such as heparin, aspirin, warfarin and otherplatelet aggregation inhibitors—that may be prescribed to treatthromboembolic disease. A second, non-limiting example of latrogeniccoagulopathy is that which is induced by excessive and/or inappropriatefluid therapy, such as that which may be induced by a blood transfusion.

In one embodiment of the current invention, haemorrhage is associatedwith haemophilia A or B. In another embodiment, haemorrhage isassociated with haemophilia A or B with acquired inhibitors. In anotherembodiment, haemorrhage is associated with von Willebrand's disease. Inanother embodiment, haemorrhage is associated with severe tissue damage.In another embodiment, haemorrhage is associated with severe trauma. Inanother embodiment, haemorrhage is associated with surgery. In anotherembodiment, haemorrhage is associated with haemorrhagic gastritis and/orenteritis. In another embodiment, the haemorrhage is profuse uterinebleeding, such as in placental abruption. In another embodiment,haemorrhage occurs in organs with a limited possibility for mechanicalhaemostasis, such as intracranially, intraaurally or intraocularly. Inanother embodiment, haemorrhage is associated with anticoagulanttherapy.

In a further embodiment, haemorrhage may be associated withthrombocytopaenia. In individuals with thrombocytopaenia, constructs ofthe current invention may be co-administered with platelets.

EMBODIMENTS

The following is a non-limiting list of embodiments of the presentinvention:

Embodiment 1

A fusion protein comprising (i) at least one tissue factor polypeptide,or biologically functional variant(s) or fragment(s) thereof, and (ii) aligand that is capable of binding (iii) a receptor, and/or a fragment orvariant thereof, wherein the receptor is present only on the surface ofactivated platelets.

Embodiment 2

The fusion protein according to embodiment 1, wherein (iii) is TLT-1 ora fragment or variant thereof.

Embodiment 3

The fusion protein according to embodiment 2, wherein (iii) is TLT-1(16-162).

Embodiment 4

The fusion protein according to embodiment 2, wherein (iii) is TLT-1(20-125).

Embodiment 5

The fusion protein according to embodiment 2, wherein (iii) is TLT-1(126-162).

Embodiment 6

The fusion protein according to any one of embodiments 1-5, wherein (i)is a single tissue factor polypeptide, or a biologically functionalvariant or fragment thereof.

Embodiment 7

The fusion protein according to any one of embodiments 1-5, wherein (i)is two tissue factor polypeptides, or biologically functional variant(s)or fragment(s) thereof.

Embodiment 8

The fusion protein according to any one of embodiments 6-7, wherein (i)is TF (1-219).

Embodiment 9

The fusion protein according to any one of embodiments 6-7, wherein (i)is sTF(6-209).

Embodiment 10

The fusion protein according to any one of embodiments 6-7, wherein (i)is sTF(1-209).

Embodiment 11

The fusion protein according to any one of embodiments 6-7, wherein (i)is sTF (1-210).

Embodiment 12

The fusion protein according to any one of embodiments 6-7, wherein (i)is sTF (1-211).

Embodiment 13

The fusion protein according to any one of embodiments 6-7, wherein (i)is sTF (1-212).

Embodiment 14

The fusion protein according to any one of embodiments 6-7, wherein (i)is sTF (1-213).

Embodiment 15

The fusion protein according to any one of embodiments 6-7, wherein (i)is sTF (1-214).

Embodiment 16

The fusion protein according to any one of embodiments 6-7, wherein (i)is sTF (1-215).

Embodiment 17

The fusion protein according to any one of embodiments 6-7, wherein (i)is sTF (1-216).

Embodiment 18

The fusion protein according to any one of embodiments 6-7, wherein (i)is sTF (1-217).

Embodiment 19

The fusion protein according to any one of embodiments 6-7, wherein (i)is sTF (1-218).

Embodiment 20

The fusion protein according to any one of embodiments 6-7, wherein (i)is sTF (1-219).

Embodiment 21

The fusion protein according to any one of embodiments 1-20, wherein(ii) is a monoclonal antibody or a fragment thereof.

Embodiment 22

The fusion protein according to embodiment 21, wherein (ii) is a Fabfragment, a F(ab′)₂ fragment, a Fab′ fragment, a Fd fragment, a Fvfragment, a ScFv fragment, a dAb fragment or an isolated complementaritydetermining region (CDR).

Embodiment 23

The fusion protein according to embodiment 22, wherein (ii) is a Fabfragment.

Embodiment 24

The fusion protein according to any one of embodiments 21-23, whereinthe epitope of (ii) comprises one or more residues selected from thegroup consisting of V17, Q18, C19, H20, Y21, R22, L23, Q24, D25, V26,K27, A28, L63, G64, G65, G66, L67, L68, G89, A90, R91, G92, P93, Q94,I95 and L96 of SEQ ID NO: 5.

Embodiment 25

The fusion protein according to any one of embodiments 21-23, wherein(ii) is an antibody, or a fragment thereof, which is capable of bindingto the same epitope as mAb 0023.

Embodiment 26

A fusion protein according to any of embodiments 24-25, wherein theheavy chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (DYFMY) of SEQ ID NO:        41, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 69 to 85 (YISNGGDSSSYPDTVKG) of        SEQ ID NO: 41, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 118 to 129 (NKNWDDYYDMDY) of SEQ        ID NO: 41, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

Embodiment 27

A fusion protein according to any of embodiments 24-26, wherein thelight chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 44 to 60 (KSSQSLLNSRTRKNYLA) of        SEQ ID NO: 42, wherein one, two, three or four of these amino        acids may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 76 to 82 (WASTRES) of SEQ ID NO:        42, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 115 to 122 (KQSYNLLT) of SEQ ID        NO: 42, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 28

A fusion protein according to any of embodiments 24-25, wherein theheavy chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (DYFMY) of SEQ ID NO:        41, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 69 to 85 (YISNGGDSSSYPDTVKG) of        SEQ ID NO: 41, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 118 to 129 (NKNWDDYYDMDY) of SEQ        ID NO: 41, wherein one, two or three of these amino acids may be        substituted by a different amino acid.        -   and wherein the light chain of (ii) comprises:    -   a CDR1 sequence of amino acids 44 to 60 (KSSQSLLNSRTRKNYLA) of        SEQ ID NO: 42, wherein one, two, three or four of these amino        acids may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 76 to 82 (WASTRES) of SEQ ID NO:        42, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 115 to 122 (KQSYNLLT) of SEQ ID        NO: 42, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 29

A fusion protein according to embodiment 28, wherein the heavy chain of(ii) comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (DYFMY) of SEQ ID NO:        41; and    -   a CDR2 sequence of amino acids 69 to 85 (YISNGGDSSSYPDTVKG) of        SEQ ID NO: 41; and    -   a CDR3 sequence of amino acids 118 to 129 (NKNWDDYYDMDY) of SEQ        ID NO: 41,        -   and wherein the light chain of (ii) comprises:    -   a CDR1 sequence of amino acids 44 to 60 (KSSQSLLNSRTRKNYLA) of        SEQ ID NO: 42; and    -   a CDR2 sequence of amino acids 76 to 82 (WASTRES) of SEQ ID NO:        42; and    -   a CDR3 sequence of amino acids 115 to 122 (KQSYNLLT) of SEQ ID        NO: 42.

Embodiment 30

The fusion protein according to any one of embodiments 21-23, whereinthe epitope of (ii) comprises one or more residues selected from thegroup consisting of L36, P37, E38, G39, C40, Q41, P42, L43, V44, S45,S46, A47, V73, T74, L75, Q76, E77, E78, D79, A80, G81, E82, Y83, G84,C85, M86, R91, G92, P93, Q94, I95, L96, H97, R98, V99, S100 and L101 ofSEQ ID NO: 5.

Embodiment 31

The fusion protein according to any one of embodiments 21-23, wherein(ii) is an antibody, or a fragment thereof, which is capable of bindingto the same epitope as mAb 0051.

Embodiment 32

A fusion protein according to any one of embodiments 30-31, wherein theheavy chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (DYSMH) of SEQ ID NO:        43, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 69 to 85 (VISTYYGDVRYNQKFKG) of        SEQ ID NO: 43, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 118 to 129 (APMITTGAWFAY) of SEQ        ID NO: 43, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

Embodiment 33

A fusion protein according to any of embodiments 30-32, wherein thelight chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 44 to 54 (KASQSVSNDVA) of SEQ ID        NO: 44, wherein one, two or three of these amino acids may be        substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 70 to 76 (YASSRYT) of SEQ ID NO:        44, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 109 to 117 (QQDYSSPYT) of SEQ ID        NO: 44, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 34

A fusion protein according to any one of embodiments 30-31, wherein theheavy chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (DYSMH) of SEQ ID NO:        43, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 69 to 85 (VISTYYGDVRYNQKFKG) of        SEQ ID NO: 43, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 118 to 129 (APMITTGAWFAY) of SEQ        ID NO: 43, wherein one, two or three of these amino acids may be        substituted by a different amino acid.        -   and wherein the light chain of (ii) comprises:    -   a CDR1 sequence of amino acids 44 to 54 (KASQSVSNDVA) of SEQ ID        NO: 44, wherein one, two or three of these amino acids may be        substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 70 to 76 (YASSRYT) of SEQ ID NO:        44, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 109 to 117 (QQDYSSPYT) of SEQ ID        NO: 44, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 35

A fusion protein according to embodiment 34, wherein the heavy chain of(ii) comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (DYSMH) of SEQ ID NO:        43; and    -   a CDR2 sequence of amino acids 69 to 85 (VISTYYGDVRYNQKFKG) of        SEQ ID NO: 43; and    -   a CDR3 sequence of amino acids 118 to 129 (APMITTGAWFAY) of SEQ        ID NO: 43;        -   and wherein the light chain of (ii) comprises:    -   a CDR1 sequence of amino acids 44 to 54 (KASQSVSNDVA) of SEQ ID        NO: 44; and    -   a CDR2 sequence of amino acids 70 to 76 (YASSRYT) of SEQ ID NO:        44; and    -   a CDR3 sequence of amino acids 109 to 117 (QQDYSSPYT) of SEQ ID        NO: 44.

Embodiment 36

The fusion protein according to any one of embodiments 21-23, whereinthe epitope of (ii) comprises one or more residues selected from thegroup consisting of V17, Q18, C19, H20, Y21, R22, L23, Q24, D25, V26,K27, A28, R91, G92, P93, Q94, I95, L96, H97, R98, V99, S100 and L101 ofSEQ ID NO: 5.

Embodiment 37

The fusion protein according to any one of embodiments 21-23, wherein(ii) is an antibody, or a fragment thereof, which is capable of bindingto the same epitope as mAb 0062.

Embodiment 38

A fusion protein according to any one of embodiments 36-37, wherein theheavy chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (SHWIE) of SEQ ID NO:        49, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 69 to 85 (EILPGSGNTNYNEKFKG) of        SEQ ID NO: 49, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 118 to 130 (GYYGLNYDWYFDV) of SEQ        ID NO: 49, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

Embodiment 39

A fusion protein according to any of embodiments 36-38, wherein thelight chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 44 to 54 (RASQDISNYLN) of SEQ ID        NO: 46, wherein one, two or three of these amino acids may be        substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 70 to 76 (YTSRLHS) of SEQ ID NO:        46, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 109 to 117 (QQDTKLPYT) of SEQ ID        NO: 46, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 40

A fusion protein according to any of embodiments 36-39, wherein theheavy chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (SHWIE) of SEQ ID NO:        49, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 69 to 85 (EILPGSGNTNYNEKFKG) of        SEQ ID NO: 49, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 118 to 130 (GYYGLNYDWYFDV) of SEQ        ID NO: 49, wherein one, two or three of these amino acids may be        substituted by a different amino acid.        -   and wherein the light chain of (ii) comprises:    -   a CDR1 sequence of amino acids 44 to 54 (RASQDISNYLN) of SEQ ID        NO: 46, wherein one, two or three of these amino acids may be        substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 70 to 76 (YTSRLHS) of SEQ ID NO:        46, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 109 to 117 (QQDTKLPYT) of SEQ ID        NO: 46, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 41

A fusion protein according to embodiment 40, wherein the heavy chain of(ii) comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (SHWIE) of SEQ ID NO:        49; and    -   a CDR2 sequence of amino acids 69 to 85 (EILPGSGNTNYNEKFKG) of        SEQ ID NO: 49; and    -   a CDR3 sequence of amino acids 118 to 130 (GYYGLNYDWYFDV) of SEQ        ID NO: 49;        -   and wherein the light chain of (ii) comprises:    -   a CDR1 sequence of amino acids 44 to 54 (RASQDISNYLN) of SEQ ID        NO: 46; and    -   a CDR2 sequence of amino acids 70 to 76 (YTSRLHS) of SEQ ID NO:        46; and    -   a CDR3 sequence of amino acids 109 to 117 (QQDTKLPYT) of SEQ ID        NO: 46.

Embodiment 42

The fusion protein according to any one of embodiments 21-23, whereinthe epitope of (ii) comprises one or more residues selected from thegroup consisting of E5, T6, H7, K8, I9, G10, S11, L12, A13, E14, N15,A16, F17, S18, D19, P20 and A21 of SEQ ID NO: 7.

Embodiment 43

The fusion protein according to embodiment 42 wherein said residues areK8, I9, G10, S11, L12, A13, N15, A16, F17, S18, D19, P20 and A21.

Embodiment 44

The fusion protein according to any one of embodiments 21-23, whereinthe epitope of (ii) comprises one or more residues selected from thegroup consisting of K118, I119, G120, S121, L122, A123, E124, N125,A126, F127 of SEQ ID NO: 6.

Embodiment 45

The fusion protein according to any one of embodiments 21-23, wherein(ii) is an antibody, or a fragment thereof, which is capable of bindingto the same epitope as mAb 0061 or mAb 0082.

Embodiment 46

A fusion protein according to any one of embodiments 42-45, wherein theheavy chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 49 to 53 (RYWMT) of SEQ ID NO:        47, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 68 to 84 (EINPDSSTINYNPSLKD) of        SEQ ID NO: 47, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 117 to 121 (GVFTS) of SEQ ID NO:        47, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

Embodiment 47

A fusion protein according to any of embodiments 42-46, wherein thelight chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 43 to 58 (RSSQSLVHRNGNTYFH) of        SEQ ID NO: 48, wherein one, two, three or four of these amino        acids may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 74 to 80 (KVSNRFS) of SEQ ID NO:        48, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 113 to 121 (SQSTHVPYT) of SEQ ID        NO: 48, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 48

A fusion protein according to any of embodiments 42-47, wherein theheavy chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 49 to 53 (RYWMT) of SEQ ID NO:        47, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 68 to 84 (EINPDSSTINYNPSLKD) of        SEQ ID NO: 47, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 117 to 121 (GVFTS) of SEQ ID NO:        47, wherein one, two or three of these amino acids may be        substituted by a different amino acid.        -   and wherein the light chain of (ii) comprises:    -   a CDR1 sequence of amino acids 43 to 58 (RSSQSLVHRNGNTYFH) of        SEQ ID NO: 48, wherein one, two, three or four of these amino        acids may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 74 to 80 (KVSNRFS) of SEQ ID NO:        48, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 113 to 121 (SQSTHVPYT) of SEQ ID        NO: 48, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 49

A fusion protein according to embodiment 48, wherein the heavy chain of(ii) comprises:

-   -   a CDR1 sequence of amino acids 49 to 53 (RYWMT) of SEQ ID NO:        47; and    -   a CDR2 sequence of amino acids 68 to 84 (EINPDSSTINYNPSLKD) of        SEQ ID NO: 47; and    -   a CDR3 sequence of amino acids 117 to 121 (GVFTS) of SEQ ID NO:        47;        -   and wherein the light chain of (ii) comprises:    -   a CDR1 sequence of amino acids 43 to 58 (RSSQSLVHRNGNTYFH) of        SEQ ID NO: 48; and    -   a CDR2 sequence of amino acids 74 to 80 (KVSNRFS) of SEQ ID NO:        48; and    -   a CDR3 sequence of amino acids 113 to 121 (SQSTHVPYT) of SEQ ID        NO: 48.

Embodiment 50

A fusion protein according to any of embodiments 42-45, wherein theheavy chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 49 to 53 (RYWMT) of SEQ ID NO:        50, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 68 to 84 (EINPDSSTINYAPSLKD) of        SEQ ID NO: 50, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 117 to 121 (GVFTS) of SEQ ID NO:        50, wherein one of these amino acids may be substituted by a        different amino acid;        -   and wherein the light chain of (ii) comprises:    -   a CDR1 sequence of amino acids 43 to 58 (RSSQSLVHRNGNTYFH) of        SEQ ID NO: 48, wherein one, two, three or four of these amino        acids may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 74 to 80 (KVSNRFS) of SEQ ID NO:        48, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 113 to 121 (SQSTHVPYT) of SEQ ID        NO: 48, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 51

A fusion protein according to embodiment 50, wherein the heavy chain of(ii) comprises:

-   -   a CDR1 sequence of amino acids 49 to 53 (RYWMT) of SEQ ID NO:        50; and    -   a CDR2 sequence of amino acids 68 to 84 (EINPDSSTINYAPSLKD) of        SEQ ID NO: 50; and    -   a CDR3 sequence of amino acids 117 to 121 (GVFTS) of SEQ ID NO:        50;        -   and wherein the light chain of (ii) comprises:    -   a CDR1 sequence of amino acids 43 to 58 (RSSQSLVHRNGNTYFH) of        SEQ ID NO: 48; and    -   a CDR2 sequence of amino acids 74 to 80 (KVSNRFS) of SEQ ID NO:        48; and    -   a CDR3 sequence of amino acids 113 to 121 (SQSTHVPYT) of SEQ ID        NO: 48.

Embodiment 52

The fusion protein according to any one of embodiments 21-23, whereinthe paratope of (ii) comprises one or more residues selected from thegroup consisting of H50, N52, Y56, H58, Y73, F79, S115, T116, V118 andY120 of the anti-TLT-1 light (L) chain (SEQ ID NO: 40), and residuesV20, F45, R49, Y50, W51, E68, T75, N77, S116, G117, V118 and T120 of theanti-TLT-1 heavy (H) chain (SEQ ID NO: 39)

Embodiment 53

The fusion protein according to any one of embodiments 21-23 and 52,wherein the epitope of (ii) comprises one or more residues selected fromthe group consisting of K133, I134, G135, S136, L137, A138, N140, A141,F142, S143, D144, P145 and A146 of SEQ ID NO: 4.

Embodiment 54

The fusion protein according to any one of embodiments 21-23, wherein(ii) is an antibody, or a fragment thereof, which is capable of bindingto the same epitope as mAb 0012.

Embodiment 55

A fusion protein according to any of embodiments 52-54, wherein theheavy chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 49 to 53 (RYWMT) of SEQ ID NO:        39, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 68 to 84 (EINPDSSTINYTPSLKD) of        SEQ ID NO: 39, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 117 to 121 (GVFTS) of SEQ ID NO:        39, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

Embodiment 56

A fusion protein according to any of embodiments 52-55, wherein thelight chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 43 to 58 (RSSQSLVHRNGNTYFH) of        SEQ ID NO: 40, wherein one, two, three or four of these amino        acids may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 74 to 80 (KVSNRFS) of SEQ ID NO:        40, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 113 to 121 (SQSTHVPYT) of SEQ ID        NO: 40, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 57

A fusion protein according to any of embodiments 52-56, wherein theheavy chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 49 to 53 (RYWMT) of SEQ ID NO:        39, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 68 to 84 (EINPDSSTINYTPSLKD) of        SEQ ID NO: 39, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 117 to 121 (GVFTS) of SEQ ID NO:        39, wherein one, two or three of these amino acids may be        substituted by a different amino acid.        -   and wherein the light chain of (ii) comprises:    -   a CDR1 sequence of amino acids 43 to 58 (RSSQSLVHRNGNTYFH) of        SEQ ID NO: 40, wherein one, two, three or four of these amino        acids may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 74 to 80 (KVSNRFS) of SEQ ID NO:        40, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 113 to 121 (SQSTHVPYT) of SEQ ID        NO: 40, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 58

A fusion protein according to embodiment 57, wherein the heavy chain of(ii) comprises:

-   -   a CDR1 sequence of amino acids 49 to 53 (RYWMT) of SEQ ID NO:        39; and    -   a CDR2 sequence of amino acids 68 to 84 (EINPDSSTINYTPSLKD) of        SEQ ID NO: 39; and    -   a CDR3 sequence of amino acids 117 to 121 (GVFTS) of SEQ ID NO:        39;        -   and wherein the light chain of (ii) comprises:    -   a CDR1 sequence of amino acids 43 to 58 (RSSQSLVHRNGNTYFH) of        SEQ ID NO: 40; and    -   a CDR2 sequence of amino acids 74 to 80 (KVSNRFS) of SEQ ID NO:        40; and    -   a CDR3 sequence of amino acids 113 to 121 (SQSTHVPYT) of SEQ ID        NO: 40.

Embodiment 59

A fusion protein according to any of embodiments 21-23, wherein theheavy chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 50 to 54 (NYWLG) of SEQ ID NO:        56, wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 69 to 85 (DIYPGGGYNKYNENFKG) of        SEQ ID NO: 56, wherein one, two, three or four of these amino        acids may be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 118 to 128 (EYGNYDYAMDS) of SEQ        ID NO: 56, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

Embodiment 60

A fusion protein according to any of embodiments 21-23 and 59, whereinthe light chain of (ii) comprises:

-   -   a CDR1 sequence of amino acids 44 to 59 (RSSRSLLHSNGNTYLC) of        SEQ ID NO: 57, wherein one, two, three or four of these amino        acids may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 75 to 81 (RMSNLAS) of SEQ ID NO:        57, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 114 to 122 (MQHLEYPFT) of SEQ ID        NO: 57, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 61

The fusion protein according to any one of embodiments 21-25, 30-31,36-37, 42-45 and 52-54, wherein (ii) is a human monoclonal antibody or afragment thereof.

Embodiment 62

The fusion protein according to any one of embodiments 1-60, wherein(ii) is a chimeric antibody or a fragment thereof.

Embodiment 63

The fusion protein according to any one of embodiments 1-60, wherein(ii) is a humanised antibody or a fragment thereof.

Embodiment 64

The fusion protein according to any one of embodiments 1-63, wherein theisotype of (ii) is IgG.

Embodiment 65

The fusion protein according to embodiment 64, wherein the isotype isIgG1, IgG2 or IgG4.

Embodiment 66

The fusion protein according to embodiment 65, wherein the isotype isIgG4.

Embodiment 67

The fusion protein according to any one of embodiments 1-66, furthercomprising a linker between (i) and (ii).

Embodiment 68

The fusion protein according to any one of embodiments 1-67, in which(ii) has a K_(D) of less than 100 nM, such as less than 10 nM.

Embodiment 69

The fusion protein according to any one of embodiments 1-68, whichstimulates FVIIa-mediated FX activation by at least 10%.

Embodiment 70

A method of targeting tissue factor, or a functional fragment thereof,to the surface of activated platelets, said method comprising thecontacting of activated platelets with a fusion protein according to anyone of embodiments 1-69.

Embodiment 71

A fusion protein according to any one of embodiments 1-67 for use as amedicament.

Embodiment 72

The fusion protein of embodiment 71 for use as a procoagulant.

Embodiment 73

A pharmaceutical formulation comprising the fusion protein according toany one of embodiments 1-69.

Embodiment 74

The fusion protein according to any one of embodiments 1-69 or thepharmaceutical formulation according to embodiment 73 for use in thetreatment of a coagulopathy.

Embodiment 75

The fusion protein according to embodiment 72, wherein said coagulopathyis haemophilia A, with or without inhibitors, and haemophilia B, with orwithout inhibitors.

Embodiment 76

A method of treating coagulopathy, comprising administering an effectiveamount of the fusion protein according to any one of embodiments 1-67 toan individual in need thereof.

Embodiment 77

The method according to embodiment 76, wherein said coagulopathy ishaemophilia A, with or without inhibitors, and haemophilia B, with orwithout inhibitors.

Embodiment 78

A polynucleotide that encodes the fusion protein according to any one ofembodiments 1-69.

Embodiment 79

An isolated cell that comprises the fusion protein according to any oneof embodiments 1-67 and/or the polynucleotide according to embodiment76.

Embodiment 80

A monoclonal antibody, or fragment thereof, that is capable of bindingTLT-1, or a fragment thereof, wherein the paratope of (ii) comprises oneor more residues selected from the group consisting of H50, N52, Y56,H58, Y73, F79, S115, T116, V118 and Y120 of the anti-TLT-1 light (L)chain (SEQ ID NO: 40), and residues V20, F45, R49, Y50, W51, E68, T75,N77, 5116, G117, V118 and T120 of the anti-TLT-1 heavy (H) chain (SEQ IDNO: 39).

Embodiment 81

A monoclonal antibody, or fragment thereof, that is capable of bindingTLT-1, or a fragment thereof, wherein the epitope of (ii) comprises oneor more residues selected from the group consisting of K133, I134, G135,S136, L137, A138, N140, A141, F142, S143, D144, P145 and A146 of SEQ IDNO: 4.

Embodiment 82

A monoclonal antibody, or fragment thereof, that is capable of bindingTLT-1, or a fragment or variant thereof, wherein the epitope of saidmonoclonal antibody comprises one or more residues selected from thegroup consisting of V17, Q18, C19, H20, Y21, R22, L23, Q24, D25, V26,K27, A28, L63, G64, G65, G66, L67, L68, G89, A90, R91, G92, P93, Q94,I95 and L96 of SEQ ID NO: 5.

Embodiment 83

A monoclonal antibody, or fragment thereof, that is capable of bindingTLT-1, or a fragment or variant thereof, wherein the epitope of saidmonoclonal antibody comprises one or more residues selected from thegroup consisting of L36, P37, E38, G39, C40, Q41, P42, L43, V44, S45,S46, A47, V73, T74, L75, Q76, E77, E78, D79, A80, G81, E82, Y83, G84,C85, M86, R91, G92, P93, Q94, I95, L96, H97, R98, V99, S100 and L101 ofSEQ ID NO: 5.

Embodiment 84

A fusion protein comprising a monoclonal antibody, or fragment thereof,that is capable of binding TLT-1, or a fragment or variant thereof,wherein the epitope of said monoclonal antibody comprises one or moreresidues selected from the group consisting of V17, Q18, C19, H20, Y21,R22, L23, Q24, D25, V26, K27, A28, R91, G92, P93, Q94, I95, L96, H97,R98, V99, S100 and L101 of SEQ ID NO: 5.

Embodiment 85

A monoclonal antibody, or fragment or variant thereof, that is capableof binding TLT-1, or a fragment thereof, wherein the epitope of saidantibody comprises one or more residues selected from the groupconsisting of E5, T6, H7, K8, I9, G10, S11, L12, A13, E14, N15, A16,F17, S18, D19, P20 and A21 of SEQ ID NO: 7.

Embodiment 86

A monoclonal antibody, or fragment or variant thereof, that is capableof binding TLT-1, or a fragment thereof, wherein said residues are K133,I134, G135, S136, L137, A138, N140, A141, F142, S143, D144, P145 andA146 of SEQ ID NO: 7.

Embodiment 87

A monoclonal antibody, or fragment or variant thereof, that is capableof binding TLT-1, or a fragment thereof, wherein the paratope of saidantibody comprises one or more residues selected from the groupconsisting of H50, N52, Y56, H58, Y73, F79, S115, T116, V118 and Y120 ofthe anti-TLT-1 light (L) chain (SEQ ID NO: 40), and residues V20, F45,R49, Y50, W51, E68, T75, N77, S116, G117, V118 and T120 of theanti-TLT-1 heavy (H) chain (SEQ ID NO: 39)

Embodiment 88

A monoclonal antibody, or fragment or variant thereof, that is capableof binding TLT-1, or a fragment thereof, wherein the epitope comprisesone or more residues selected from the group consisting of K133, I134,G135, S136, L137, A138, N140, A141, F142, S143, D144, P145 and A146 ofSEQ ID NO: 4.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references cited throughout this application areexpressly incorporated herein by reference.

EXAMPLES Example 1

Cloning and Expression of hTLT-1 ECD-His Antigen.

Nucleotide sequences encoding the extracellular domain of human TLT-1(hTLT-1) (FIG. 1) together with a C-terminal His-6 tag were PCRamplified with a forward primer containing a HindIII recognition sitetogether with a kozak sequence, and a reverse primer containing a stopcodon and an EcoRI recognition site (FIG. 2). The HindIII- and EcoRIdigested PCR fragment was inserted into the HindIII- and EcoRI sites ofa pTT-based expression vector. The pTT vector is essentially describedin Durocher, Y. et al., (2002) Nucleic Acid Res, 30: E9. The resultingexpression plasmid was designated pTT-hTLT-1 ECD-His. pTT-hTLT-1 ECD-Hiswas transfected into HEK293-6E suspension cells in order to transientlyexpress hTLT-1 ECD-His. HEK293-6E cells were grown in FREESTYLE HEK293MEDIUM (animal origin-free, chemically defined, protein-freemedium)(GIBCO, cat. no. 12338-018) supplemented with 1% P/S (GIBCO cat.no. 15140-122), 0.1% PLURONIC (clock copolymers based on ethylene oxideand propylene oxide) (GIBCO, cat. no. 24040-032) and 25 ug/mL GENETICIN(aminoglycoside selective agent) (GIBCO, cat. no. 10131-019) and cellswere transfected at a cell density of 1 mill/mL using 293FECTIN(cationic lipid-based formulation for transfecting DNA into eukaryoticcells) (Invitrogen, cat. no. 12347-019). For each liter of HEK293-6Ecells, the transfection was performed by diluting 1 mg of pTT-hTLT-1ECD-His DNA into 30 mL OPTIMEM (reduced-serum media) (dilution A) and bydiluting 1 mL 293FECTIN (cationic lipid-based formulation fortransfecting DNA into eukaryotic cells) into 30 mL OPTIMEM(reduced-serum media) (GIBCO, cat. no. 51985-026, dilution B). DilutionA and B were mixed and incubated at room temperature for 30 minutes. Thetransfection mix was hereafter added to the HEK293-6E cells and cellswere incubated at 37° C. in a humified incubator with orbital rotation(125 rpm). Seven days post-transfection, cells were removed bycentrifugation and the resulting hTLT-1 ECD-His containing supernatantswere sterile-filtrated prior to purification.

Example 2

Purification and Characterisation of hTLT1 ECD-His Protein.

Purification of the hTLT1 ECD-His protein was conducted as a 2-stepprocess composed of 1) His-affinity chromatography using theCobalt-loaded resin TALON (Clontech, cat. no. 635506) and 2)anion-exchange chromatography using the fine-particle resin Source 15Q(GE Healthcare, cat. no. 17-0947). The purifications were conductedusing an ÄktaExplorer chromatography system (GE Healthcare, cat. no.18-1112-41). The buffer systems used for the first purification step wasan equilibration buffer composed of 20 mM Hepes, pH 7.0, 150 mM NaCl, awash buffer composed of 20 mM Hepes, pH 7.0, 0.5 M NaCl and an elutionbuffer composed of 20 mM Hepes, pH 7.0, 150 mM Imidazole. The cellsupernatant was applied directly without any adjustments onto apre-equilibrated TALON column. The column was washed with 20 columnvolumes of equilibration buffer, 20 column volumes of wash buffer andlast with 20 column volumes of equilibration buffer. The protein waseluted isocratically in approx. 5 column volumes of elution buffer. Themolecular mass of the eluted protein was ana-lysed usingSDS-PAGE/Coomassie NUPAGE (Bis-Tris gel) 4-12% Bis-Tris gels(Invitrogen, cat. no. NP0321BOX) and Matrix Assisted Laser DesorptionIonisation Time-of-Flight Mass Spectrometry (MALDI-TOF MS) setup on aMicro-flex system (Bruker Daltonics). Here, two distinct protein masseswere observed of approx. 16.7 and 33.4 kDa of almost equal amounts. Theobserved masses corresponded to monomer and dimer forms of hTLT1ECD-His. Reducing the protein resulted in complete abolishment of the33.4 kDa protein, while intensifying the 16.7 kDa protein as judged froma SDS-PAGE/Coomassie analysis. Thus, the hTLT-1 ECD-His proteincontained an interlinked C-C dimer. To segregate monomer from dimer, asecond purification step was employed. The buffer systems used for thispurification step was an equilibration buffer composed of 50 mM Hepes,pH 8.0 and an elution buffer composed of 50 mM Hepes, pH 8.0, 1 M NaCl.The sample was adjusted to a pH of 8.0 using 1 M NaOH and then dilutedto a conductivity of approx. 10 mS/cm. The protein was applied to apre-equilibrated Source 15Q column, washed with 5 column volumes ofequilibration and eluted using 0-100% elution buffer over 20 columnvolumes. Based on UV280 monitoring, two peaks were apparent with almostbase-line separa-tion. Analyzing fractions over the two peaks usingSDS-PAGE/Coomassie, MALDI-TOF MS and Dynamic Light-Scattering (DLS)using a Dynapro instrument (Wyatt Technology) analyses showed thepresence of monomer hTLT-1 ECD-His protein in the peak eluting first andCys-Cys dimer in the peak eluting second. A pool was prepared containingthe monomer hTLT-1 ECD-His protein only. The final protein integrity wasanalyzed based on a Size-Exclusion High-Performance LiquidChromatographic (SEC-HPLC) method setup on an Agilent LC 1100/1200system and using a BIOSEP (column for separation biomolecules)-SEC-S3000300×7.8 mm column (Phenomenex, cat. no. 00H-2146-K0) and a runningbuffer composed of 200 mM NaPhosphate pH 6.9, 300 mM NaCl and 10%isopropanol. The protein eluted as a single symmetric peak at aretention time of approx. 9.9 min at a flow rate of 1 ml/min.

A batch of hTLT-1 ECD-His was prepared for an immunization study forproduction of monoclonal anti-TLT1 antibodies. Thus, the protein wasdialyzed into an isotonic PBS buffer using a SLIDE-A-LYZER DialysisCassette 10 kDa MWCO (Pierce, cat. no. 66453). To measure the finalprotein concentration, a NANODROP spectropho-tometer (Thermo Scientific)was used together with an extinction coefficient of 0.55.

Example 3

Preparation of Monoclonal TLT-1 Antibodies.

RBF mice were immunized by injecting 50 μg of hTLT-1 ECD-His. FCAsubcutaneously followed by two injections with 20 μg of hTLT-1 ECD-Hisin FIA. High responder mice were boosted intravenously with 25 μg ofhTLT-1 ECD-His and the spleens were harvested after 3 days. Spleen cellswere fused with the myeloma Fox cell line. Supernatants were screenedfor hTLT-1 specific antibody production in a specific ELISA and in aFACS assay utilizing hTLT-1- or Mock-transfected CHO cells as positiveand negative target cells, respectively. A secondary screen was done onresting versus dual agonistic activated platelets of human, cynomolgousmonkey, dog, rabbit or mouse origin.

Example 4

Cloning and Sequencing of antiTLT-1 mAb LC and HC cDNAs from Hybridoma.

Total RNA was extracted from four different anti-TLT-1 mAb expressinghybridoma designated: 0012Hyb (a.k.a. 2F105/2F105A3), 0023Hyb, 0051Hyband 0052Hyb. The RNA was extracted from hybridoma cells using RNEASY(kit for purifying total RNA from cells, tissues, and yeast) mini kit(Qiagen, cat. no. 74106) and an aliquot of the resulting RNA was used astemplate for first-stranded cDNA synthesis using SMART RACE cDNAAmplification kit (Clontech, cat. no. 634914) following the instructionof the manufacturer for 5′ RACE and using 5′ RACE CDS primer A togetherwith SMART II A RNA oligonucleotide. The light chain (LC) and heavychain (HC) coding region cDNAs from each of the four anti-TLT-1hybridomas were hereafter PCR amplified using UPM forward primer mixtogether with either a mouse LC,kappa specific reverse primer (reverseprimer number 339, 348, or 610) or together with a reverse primerrecognizing mouse IgG1, IgG2a, IgG2b or IgG3 sequences (reverse primernumber 341, 347, 613, 614, 615, or 616, primer sequences are shown inseq no 70-155). The PCR reactions were performed using PHUSION PCR mix(FinnZymes, cat no.: F-531L). The resulting PCR fragments were clonedusing ZERO BLUNT Topo PCR cloning kit for sequencing (Invitrogen, cat.no. K287540) and sequenced. The variable domain sequences for 0012LC andHC are shown in FIG. 3.

Example 5

Development of pTT-0012HC, pTT-0023HC, pTT-0051HC and pTT-0052HCExpression Constructs.

The HC variable domain (V_(H)) encoding DNA sequences isolated from eachof the four different antiTLT-1 hybridomas were PCR amplified withforward primers containing a HinDIII restriction enzyme site and reverseprimers containing a NheI restriction enzyme site for cloning purposes.The 0012V_(H), 0023V_(H), 0051V_(H) and 0052V_(H) DNA sequences were PCRamplified using PHUSION PCR mix (FinnZymes, cat No. F-531L) with thefollowing primer number pairs: 490 (forward)+491 (reverse), 546(forward)+547 (reverse), 627 (forward)+628 (reverse), and 617(forward)+618 (reverse, primer sequences are shown in seq no 70-155),respectively, and inserted into the HinDIII and NheI restriction enzymesites of a pTT based vector (FIG. 22+23) designated, pTT-hIgG4,containing the constant region encoding sequences for human IgG4 HC (ieCH1-hinge-CH2-CH3). The pTT vector is essentially described in Durocher,Y. et al., (2002) Nucleic Acid Res, 30: E9 (FIG. 22). The resultingvectors were designated pTT-0012HC (FIG. 5), pTT-0023HC, pTT-0051HC, andpTT-0052HC. The antiTLT-1 HC amino acid sequences encoded by theexpression vectors are shown (Seq. ID no.: 0012HC: 39, 0023HC: 41,0051HC: 43, 0052HC: 45).

Example 6

Development of pTT-0012LC, pTT-0023LC, pTT-0051LC and pTT-0052LCExpression Constructs.

The LC variable domain (V_(L)) encoding DNA sequences isolated from eachof the four different antiTLT-1 hybridomas were PCR amplified withforward primers containing a HinDIII restriction enzyme site and reverseprimers containing a BsiWI restriction enzyme site for cloning purposes.The 0012V_(L), 0023V_(L), 0051V_(L) and 0052V_(L) DNA sequences were PCRamplified with the following primers number pairs: 493 (forward)+495(reverse), 548 (forward)+549 (reverse), 492 (forward)+494 (reverse), and619 (forward)+620 (reverse primer sequences are shown in seq no 70-155),respectively, and inserted into the HinDIII and BsiWI restriction enzymesites of a pTT-based vector designated, pTT-hLC, Kappa, containing theconstant region encoding sequences for human LC, kappa (FIG. 24). Theresulting vectors were designated pTT-0012LC, pTT-0023LC, pTT-0051LC,and pTT-0052LC. The antiTLT-1 LC amino acid sequence encoded by theexpression vectors are shown in (Seq. ID no.: 0012LC: 40, 0023LC: 42,0051LC: 44, 0052LC: 46).

Example 7

Development of pTT-0012HC.T60N, pTT-0012HC.T60A, pTT-0012LC.C36A andpTT-0052HC.C91Y.

The 0012V_(H) amino acid sequence contains a potential N-linkedglycosylation site (T60, kabat numbering) and the 0012V_(L) and the0052V_(H) amino acid sequences each contain an unpaired Cys (C36 andC91, respectively, kabat numbering). Expression vectors encoding0012HC.T60N or 0012HC.T60A or 0012LC.C36A or 0052HC.C91Y were developedusing site directed mutagenesis (QUICHANGE II, Stratagene, Catalognumber 20523-5) following the instructions of the manufacturer. Thesite-directed mutagenesis reactions were performed using a) pTT-0012HCDNA as template and primer numbers 682 (forward)+683 (reverse) forpTT-0012HC.T60N, b) pTT-0012HC DNA as template and primer numbers 688(forward)+689 (reverse) for pTT-0012HC.T60A, c) pTT-0012LC DNA astemplate and primer numbers 598 (forward)+599 (reverse) forpTT-0012LC.C36A, d) pTT-0052HC DNA as template and the following primernumbers 684 (forward)+685 (reverse, primer sequences are shown in seq no70-155) for pTT-0052HC.C91Y. The resulting expression vectors weresequenced in order to verify DNA sequences. The antiTLT-1 HC and LCamino acid sequence encoded by the pTT-0012HC.T60N, pTT-0012HC.T60A andpTT-0012LC.C36A expression vectors are shown in (Seq. ID no.:0012HC.T60N: 47, 0012HC.T60A: 50, 0012LC.C36A: 48).

Example 8

Development of pTT-0012LC-HPC4, pTT-0012LC.C36A-HPC4, pTT-0023LC-HPC4,pTT-0051LC-HPC4 and pTT-0052LC-HPC4 Expression Constructs.

V_(L) encoding DNA sequences isolated from each of the four differentantiTLT-1 hybridomas were PCR amplified with forward primers containinga HinDIII restriction enzyme site and reverse primers containing a BsiWIrestriction enzyme site for cloning purposes. 0012V_(L), 0012V_(L).C36A,0023V_(L), 0051V_(L) and 0052V_(L) DNA sequences were PCR amplified withthe following primer numbers: 493 (forward)+495 (reverse), 548(forward)+549 (reverse), 492 (forward)+494 (reverse), and 619(forward)+620 (reverse, primer sequences are shown in Seq. ID no.70-155) respectively, using PHUSION PCR mix (FinnZymes, cat No. F-531L).The human C_(L),kappa encoding sequence was PCR amplified with forwardprimer number 486 and reverse primer number 485. Forward primer number486 contains a BsiWI restriction enzyme site and reverse primer 485encodes a HPC4 tag (Seq. ID no.: 69) followed by a stop codon andcontains a 3′ flanking EcoRI site for cloning purposes. The PCR reactionwas performed using PHUSION PCR mix (FinnZymes, cat No. F-531L).HindIII+BsiWI digested 0012V_(L) PCR fragment was mixed with BsiWI+EcoRIdigested human C_(L),kappa-HPC4 PCR fragment and inserted into theHinDIII+EcoRI sites of a pTT-based expression vector resulting inpTT-0012LC-HPC4 (FIG. 4). In order to develop corresponding expressionvectors encoding the LC-HPC4 version of the remaining three antiTLT-1 LCsequences, the 0012V_(L) sequence in pTT-0012LC-HPC4 was excised withHinDIII+BsiWI and replaced with HinDIII+BsiWI digested 0023V_(L),0051V_(L), 0052V_(L) and 0012V_(L).C36A PCR fragments. The resultingfour expression vectors were designated: pTT-0023LC-HPC4,pTT-0051LC-HPC4, pTT-0052LC-HPC4 and pTT-0012LC.C36A.HPC4.

Example 9

Development of pTT-0012V_(H)-CH1, pTT-0012V_(H)-CH1-HPC4,pTT-0023V_(H)-CH1, pTT-0023V_(H)-CH1-HPC4, pTT-0051V_(H)-CH1,pTT-0051V_(H)-CH1-HPC4, pTT-0052V_(H)-CH1 and pTT-0052V_(H)-CH1-HPC4Expression Constructs.

The 0012V_(H), 0023V_(H), 0051V_(H), and 0052V_(H) sequences isolatedfrom 0012Hyb, 0023Hyb, 0051Hyb, 0052Hyb were PCR amplified with primernumbers: 490 (forward)+491 (reverse), 546 (forward)+547 (reverse), 627(forward)+628 (reverse), 617 (forward)+618 (reverse, primer sequencesare shown in seq no 70-155), respectively, using PHUSION PCR mix(FinnZymes, cat No. F-531L). All forward primers (490, 546, 627, and617) contained a HinDIII site and all reverse primers (491, 547, 628,and 618) contained a NheI site for cloning purposes. The human IgG₄ CH1region was PCR amplified either with primer numbers: 489 (forward)+488(reverse), or primer numbers 489 (forward)+487 (reverse). Forward primernumber 489 contained a NheI site, the 488 reverse primer numbercontained a stop codon and an EcoRI site, and the 487 reverse primernumber contained an HPC4 tag encoding sequence, a stop codon followed byan EcoRI site for cloning purposes. HinDIII+NheI digested 0012V_(H) PCRfragment was combined with either NheI+EcoRI digested human IgG₄ CH1 PCRfragment or with NheI+EcoRI digested human IgG₄ CH1-HPC4 PCR fragmentand cloned into the HindIII+EcoRI sites for a pTT based vector. Theresulting vectors were designated pTT-0012V_(H)-CH1 andpTT-0012V_(H)-CH1-HPC4, respectively. Subsequently, the V_(H) domains ofpTT-0012V_(H)-CH1 and of pTT-0012V_(H)-CH1-HPC4 were excised byHindIII+NheI digestion and HinDIII+NheI digested 0197-0000-0023V_(H),0197-0000-0051V_(H) and 0197-0000-0052V_(H) PCR fragments were inserted.The resulting expression vectors were designated: pTT-0023V_(H)-CH1,pTT-0023V_(H)-CH1-HPC4, pTT-0051V_(H)-CH1, pTT-0051V_(H)-CH1-HPC4,pTT-0052V_(H)-CH1, and pTT-0052V_(H)-CH1-HPC4.

Example 10

Development of pTT-0012V_(H).T60N-CH1 and pTT-0012V_(H).T60N-CH1-HPC4Expression Constructs.

The 0012V_(H).T60N-CH1 sequence (including the signal peptide encodingsequence) was PCR amplified from pTT-0012HC.T60N using PHUSION PCR mix(FinnZymes, cat No. F-531L) and using forward primer number 572containing a HinDIII restriction enzyme site and reverse primer number488 containing a EcoRI site for cloning purposes or reverse primer 487containing a HPC4 tag encoding sequence together with a EcoRI site forcloning purposes (primer sequences are shown in seq no 70-155). Theresulting PCR fragments were digested with HinDIII+EcoRI and insertedinto the HinDIII+EcoRI sites of a pTT based vector. The resultingexpression vectors were designated pTT-0012V_(H).T60N-CH1 andpTT-0012V_(H).T60N-CH1-HPC4.

Example 11

Development of pTT-L4a-hTF.1-219 and pTT-hTF.1-219-L4b ExpressionConstructs.

An expression construct was made encoding an N-terminal 17 amino acidGly-Ser linker (L4a: GSGGGGSGGGGS GGGGS, Seq. ID no. 61) and theextracellular domain of human tissue factor excluding the signal peptideencoding sequence (hTF.1-219, Seq. ID no. 14). At first, the hTF.1-219cDNA sequence was PCR amplified using PHUSION PCR mix (FinnZymes, catNo. F-531L) and using primer number 466 (forward) containing L4aencoding DNA sequence and reverse primer 449 (primer sequences are shownin seq no 70-155) resulting in the L4a-hTF.1-219 PCR fragment. A secondPCR amplification step was performed with primer number 483 (forward)and 449 (reverse) using the first PCR fragment as template. The secondPCR step was done in order to incorporate both HinDIII and EcoRI sitesinto the PCR fragment for cloning purposes. The resulting PCR fragmentencoded L4a-hTF.1-219 and contained a BamHI site as part of the Gly-Serlinker (underlined sequence in L4a) for future cloning purposes. The DNAfragment was inserted into the HinDIII and EcoRI sites of a pTT-basedvector and the resulting vector was designated pTT-L4a-hTF. 1-219 (FIG.25).

An expression construct was made encoding the extracellular domain ofhuman tissue factor including the signal peptide encoding sequence and aC-terminal 17 amino acid Gly-Ser linker (L4b: GGGGSGGGGSGGGGS GS, Seq.ID no. 62). At first, hTF.1-219 was PCR amplified using PHUSION PCR mix(FinnZymes, cat No. F-531L) and using primer number 448 (forward) and467 (reverse, primer sequences are shown in seq. ID no 70-155) resultingin a DNA fragment encoding hTF.1-219-L4b (Seq. ID no. 16(AA1-251)+62). Asecond PCR amplification step was performed with primer number 448(forward) and 484 (reverse) using the first PCR fragment as template.The second PCR step was done in order to incorporate both HinDIII andEcoRI sites into the PCR fragment for cloning purposes. The resultinghTF.1-219-L4b encoding PCR fragment contained a BamHI site as part ofthe Gly-Ser linker (underlined sequence in L4b) for future cloningpurposes. The DNA fragment was inserted into the HinDIII and EcoRI sitesof a pTT-based vector and the resulting vector was designatedpTT-hTF.1-219-L4b (FIG. 26).

Example 12

Development of pTT-0012LC-L4a-hTF.1-219, pTT-hTF.1-219-L4b-0012LCExpression and pTT-0012LC.C36A-L4a-hTF.1-219 Constructs.

The 0012LC cDNA (including the signal peptide encoding sequence) was PCRamplified from pTT-0012LC using PHUSION PCR mix (FinnZymes, cat No.F-531L) and using forward primer number 493 and reverse primer number552. The forward primer 493 inserted a 5′end HinDIII restriction enzymesite and the reverse primer 552 inserted a 3′end BamHI restrictionenzyme site for cloning purposes (primer sequences are shown in seq no70-155). The resulting 0012LC PCR fragment was inserted into theHinDIII+BamHI sites of pTT-L4a-hTF.1-219 resulting in the0012LC-L4a-hTF.1-219 encoding expression vector designatedpTT-0012LC-L4a-hTF.1-219.

The 0012LC cDNA (excluding the signal peptide encoding sequence) was PCRamplified from pTT-0012LC using PHUSION PCR mix (FinnZymes, cat No.F-531L) and using forward primer number 551 and reverse primer number98. The forward primer 551 inserted a 5′end BamHI restriction enzymesite and the reverse primer 98 inserted a 3′end EcoRI restriction enzymesite for cloning purposes (primer sequences are shown in seq no 70-155).The resulting 0012LC PCR fragment was inserted into the BamHI+EcoRIsites of pTT-hTF.1-219-L4b resulting in the hTF.1-219-L4b-0012LCencoding expression vector designated pTT-hTF. 1-219-L4b-0012LC.

The 0012V_(L).C36A cDNA sequence was excised from pTT-0012LC.C36A usingthe HinDIII and BsiWI restriction enzymes and the resulting DNA fragmentwas inserted into the HinDIII+BsiWI restriction enzyme sites of thepTT-0012LC-L4a-hTF.1-219 plasmid i.e. replacing the 0012V_(L) sequence.The resulting expression plasmid was designated pTT-0012LC.C36A-L4a-hTF.1-219 (also designated 0061LC-L4a-hTF. 1-219).

Example 13

Development of pTT-0012HC-L4a-hTF.1-219 and pTT-hTF.1-219-L4b-0012HCExpression Constructs.

The 0012HC sequence (including the signal peptide encoding sequence) wasPCR amplified from pTT-0012HC using PHUSION PCR mix (FinnZymes, cat No.F-531L) and using forward primer number 490 and reverse primer number513. The forward primer 490 inserted a 5′end HinDIII restriction enzymesite and reverse primer 513 inserted a 3′end BamHI restriction enzymesite for cloning purposes. The 0012HC PCR fragment was inserted into theHinDIII+BamHI sites of pTT-L4a-hTF.1-219 resulting in the0012HC-L4a-hTF.1-219 expression construct designatedpTT-0012HC-L4a-hTF.1-219.

The 0012HC sequence (excluding the signal peptide encoding sequence) wasPCR amplified from pTT-0012HC using PHUSION PCR mix (FinnZymes, cat No.F-531L) and using forward primer number 512 and reverse primer number100. The forward primer 512 inserted a 5′end BamHI restriction enzymesite and reverse primer 100 inserted a 3′end EcoRI restriction enzymesite for cloning purposes (primer sequences are shown in seq no 70-155).The 0012HC PCR fragment was inserted into the BamHI+EcoRI sites ofpTT-hTF.1-219-L4b resulting in the hTF.1-219-L4b-0012HC expressionconstruct designated pTT-hTF.1-219-L4b-0012HC.

Example 14

Development of pTT-0012V_(H)-CH1-L4a-hTF.1-219 andpTT-hTF.1-219-L4b-0012V_(H)-CH1 Constructs.

The 0012V_(H)-CH1 encoding DNA sequence (including the signal peptideencoding sequence) was PCR amplified from pTT-0012HC using PHUSION PCRmix (FinnZymes, cat No. F-531L) and using forward primer number 490 andreverse primer number 514 (primer sequences are shown in Seq. ID no70-155). The forward primer 490 inserted a 5′end HinDIII restrictionenzyme site and the reverse primer 514 inserted a 3′end BamHIrestriction enzyme site for cloning purposes. The 0012V_(H)-CH1 PCRfragment was inserted into the HinDIII+BamHI sites of pTT-L4a-hTF.1-219resulting in the 0012V_(H)-CH1-L4a-hTF.1-219 expression constructdesignated pTT-0012V_(H)-CH1-L4a-hTF.1-219 (FIG. 6).

The 0012V_(H)-CH1 encoding DNA sequence (excluding the signal peptideencoding sequence) was PCR amplified from pTT-0012HC using forwardprimer number 512 and reverse primer number 488. The forward primer 512inserted a 5′end BamHI restriction enzyme site and the reverse primer488 inserted a stop codon and a 3′end EcoRI restriction enzyme site forcloning purposes (primer sequences are shown in seq no 70-155). The0012V_(H)-CH1 PCR fragment was inserted into the BamHI+EcoRI sites ofpTT-hTF.1-219-L4b resulting in the hTF.1-219-L4b-0012V_(H)-CH1expression construct designated pTT-hTF.1-219-L4b-0012V_(H)-CH1.

Example 15

Development of pTT-0012LC-TF with Different Linker Length: L0 (NoLinker), L1 (2GS), L2 (7GS), L3 (12GS), L5 (22GS), L6 (27GS), L7 (32GS),L8 (37GS), L9 (42GS).

The 0012LC cDNA sequence (including the signal peptide encodingsequence) was PCR amplified using PHUSION PCR mix (FinnZymes, cat No.F-531L) and using forward primer number 574 containing a 5′end HinDIIIsite and reverse primers containing 3′end sequences encoding either GSlinker length of 5 GS+a BamHI site (reverse primer number 590), 10GS+aBamHI site (reverse primer number 585), 15GS (reverse primer number583)+a BamHI site, 20GS+a BamHI site (reverse primer number 584), 25GS+aBamHI site (reverse primer number 591, primer sequences are shown in seqno 70-155). The resulting 0012LC PCR fragments were digested withHinDIII+BamHI and inserted into pTT-L4a-hTF.1-219 resulting inexpression vectors designated pTT-0012LC-L5-hTF.1-219 (22GS linker),pTT-0012LC-L6-hTF.1-219 (27GS linker), pTT-0012LC-L7-hTF.1-219 (32GSlinker), pTT-0012LC-L8-hTF.1-219 (37GS linker) andpTT-0012LC-L9-hTF.1-219 (42GS linker).

The hTF.1-219 cDNA sequence (excluding the signal peptide encodingsequence) was PCR amplified using PHUSION PCR mix (FinnZymes, cat No.F-531L) and using forward primer number 586, containing a BamHI site(2GS linker) following by a 10GS linker encoding sequence, or forwardprimer number 699 containing a BamHI site (2GS linker) followed by a 5GSlinker encoding sequence or forward primer number 700 containing a BamHIsite (2GS linker) together with reverse primer number 449 containing aEcoRI restriction enzyme site for cloning purposes (primer sequences areshown in seq no 70-155). The resulting PCR fragments were digested withBamHI+EcoRI and inserted into the BamHI+EcoRI sites ofpTT-0012LC-L4a-hTF.1-219 i.e. replacing the L4a-hTF.1-219 sequence. Theresulting expression vectors were designated: pTT-0012LC-L3-hTF.1-219(12GS linker), pTT-0012LC-L2-hTF.1-219 (7GS linker) orpTT-0012LC-L1-hTF.1-219 (2GS linker).

The 0012LC cDNA sequence (including the signal peptide encodingsequence) was PCR amplified using PHUSION PCR mix (FinnZymes, cat No.F-531L) and using forward primer number 574 containing a 5′end HinDIIIsite and the reverse primer number 704 containing sequences from the5′end of hTF.1-219. The hTF.1-219 cDNA sequence (excluding the signalpeptide encoding sequence) was PCR amplified using PHUSION PCR mix(FinnZymes, cat No. F-531L) and using forward primer number 703containing sequences from the 3′end of 0012LC and reverse primer number449 containing a 3′end EcoRI site. The 0012LC and hTF.1-219 PCRfragments were combined and a second PCR step (overlap PCR) wasperformed using forward primer number 574 and reverse primer number 449(primer sequences are shown in seq no 70-155). The resulting PCRfragment was inserted into the HinDIII+EcoRI sites of a pTT-basedexpression vector resulting in pTT-0012LC-L0-hTF.1-219 encoding a0012LC-hTF.1-219 fusion protein without any linker sequences.

Example 16

Development of pTT-0023LC-TF with Different Linker Length: L3 (12GS),L4a (17GS), L5 (22GS), L6 (27GS), L7 (32GS), L8 (37GS), L9 (42GS).

The 0023V_(L) cDNA sequence (including the signal peptide encodingsequence) was excised from pTT-0023LC using the HinDIII+BsiWIrestriction enzymes and inserted into the HinDIII+BsiWI restrictionenzyme sites of: pTT-0012LC-L3-hTF.1-219 (12GS linker),pTT-0012LC-L4a-hTF.1-219 (17GS linker), pTT-0012LC-L5-hTF.1-219 (22GSlinker), pTT-0012LC-L6-hTF.1-219 (27GS linker), pTT-0012LC-L7-hTF.1-219(32GS linker), pTT-0012LC-L8-hTF.1-219 (37GS linker) andpTT-0012LC-L9-hTF.1-219 (42GS linker) i.e. replacing the 0012V_(L) cDNAsequence with 0023V_(L). The resulting expression vectors weredesignated: pTT-0023LC-L3-hTF.1-219 (12GS linker),pTT-0023LC-L4a-hTF.1-219 (17GS linker), pTT-0023LC-L5-hTF.1-219 (22GSlinker), pTT-0023LC-L6-hTF.1-219 (27GS linker), pTT-0023LC-L7-hTF.1-219(32GS linker), pTT-0023LC-L8-hTF.1-219 (37GS linker) andpTT-0023LC-L9-hTF.1-219 (42GS linker).

Example 17

Development of pTT-0023HC-TF with Different Linker Length: L1 (2GS), L2(7GS), L3 (12GS), L4a (17GS), L5 (22GS), L6 (27GS), L7 (32GS), L8(37GS), L9 (42GS).

The 0023HC cDNA sequence (including the signal peptide encodingsequence) was PCR amplified using PHUSION PCR mix (FinnZymes, cat No.F-531L) and using forward primer number 546 containing a 5′-end HindIIIrestriction enzyme site and 1) reverse primer number 592 containing a5GS encoding sequence together with a BamHI site or 2) reverse primernumber 589 containing a 10GS encoding sequence together with a BamHIsite or 3) reverse primer number 587 containing a 15GS encoding sequencetogether with a BamHI site or 4) reverse primer number 588 containing a20GS encoding sequence together with a BamHI site or 5) reverse primernumber 593 containing a 25GS encoding sequence together with a BamHIsite (primer sequences are shown in seq no 70-155). The resulting PCRfragments were digested with HinDIII+BamHI and inserted into theHinDIII+BamHI restriction enzyme sites of pTT-0012LC-L4a-hTF.1-219 i.e.replacing the 0012LC encoding sequence. The resulting expression vectorswere designated: pTT-0023HC-L5-hTF.1-219 (22GS linker),pTT-0023HC-L6-hTF.1-219 (27GS linker), pTT-0023HC-L7-hTF.1-219 (32GSlinker), pTT-0023HC-L8-hTF.1-219 (37GS linker), pTT-0023HC-L9-hTF.1-219(42GS linker).

The hTF.1-219 cDNA sequence (excluding the hTF signal peptide sequence)was PCR amplified using PHUSION PCR mix (FinnZymes, cat No. F-531L) andusing a) forward primer number 586 containing a BamHI restriction enzymesite and a 10GS encoding sequence or b) forward primer number 699containing a BamHI restriction enzyme site and a 5GS encoding sequenceor forward primer number 700 containing a BamHI restriction enzyme sitetogether with reverse primer number 449 containing an EcoRI restrictionenzyme site for cloning purposes (primer sequences are shown in seq no70-155). The resulting PCR fragments were digested with BamHI+EcoRI andinserted into the BamHI+EcoRI sites of pTT-0023HC-L4a-hTF.1-219 i.e.replacing the L4a-hTF.1-219 sequence with L3-hTF.1-219 or L2-hTF.1-219or L1-hTF.1-219 cDNA sequence. The resulting expression vectors weredesignated pTT-0023HC-L3-hTF.1-219 (12GS linker),pTT-0023HC-L2-hTF.1-219 (7GS linker) and pTT-0023HC-L1-hTF.1-219 (2GSlinker), respectively. The 0023V_(H) DNA sequence was excised frompTT-0023HC using HinDIII+NheI and inserted into the HinDIII+NheI sitesof pTT-0012HC-L4a-hTF.1-219 i.e. replacing the 0012V_(H) DNA sequence.The resulting expression vector was designated pTT-0023HC-L4a-hTF.1-219.

Example 18

Development of pTT-0012HC-TF with Different Linker Length: L0 (NoLinker), L1 (2GS), L2 (7GS), L3 (12GS), L5 (22GS), L6 (27GS), L7 (32GS),L8 (37GS), L9 (42GS).

The 0012V_(H) encoding cDNA sequence was excised from pTT-0012HC usingHinDIII+NheI and the resulting cDNA fragment was inserted intopTT-0023HC-L1-hTF.1-219, pTT-0023HC-L2-hTF.1-219, pTT-0023HC-L3-hTF.1-219, pTT-0023HC-L5-hTF.1-219, pTT-0023HC-L6-hTF.1-219,pTT-0023HC-L7-hTF. 1-219, pTT-0023HC-L8-hTF.1-219 orpTT-0023HC-L9-hTF.1-219 i.e. replacing 0023V_(H). The resultingexpression vectors were designated: 1) pTT-0012HC-L1-hTF. 1-219,pTT-0012HC-L2-hTF.1-219, pTT-0012HC-L3-hTF.1-219,pTT-0012HC-L5-hTF.1-219, pTT-0012HC-L6-hTF.1-219,pTT-0012HC-L7-hTF.1-219, pTT-0012HC-L8-hTF.1-219 or pTT-0012HC-L9-hTF.1-219.

0012HC encoding cDNA (including the signal peptide sequence) was PCRamplified using PHUSION PCR mix (FinnZymes, cat No. F-531L) and usingforward primer number 490 containing a 5′end HinDIII restriction enzymesite and reverse primer number 801 containing a part of the 5′end cDNAsequence encoding hTF.1-219. The hTF.1-219 cDNA sequence (excluding thesignal peptide encoding sequence) was PCR amplified using forward primernumber 800 containing a 5′end sequence encoding the 3′end part of 0012HCcDNA and reverse primer number 449 containing an EcoRI site for cloningpurposes (primer sequences are shown in seq no 70-155). The resultingtwo PCR fragments were combined and used as template in a second PCRstep (overlapping PCR) using forward primer number 490 and reverseprimer number 449. The PCR fragment obtained was digested withHinDIII+EcoRI and inserted into a pTT based expression vector. Theresulting expression vector was designated pTT-0012-L0-hTF.1-219 andencoded a 0012HC-TF fusion protein without linker sequence.

Example 19

Development of pTT-0012V_(H)-CH1-L10-hTF.1-219,pTT-0012V_(H).T60N-CH1-L10-hTF.1-219 and pTT-0012V_(H).T60A-CH1-L10-hTF.1-219.

The 0012V_(H)-CH1-hinge cDNA sequence (including the signal peptideencoding sequence) was PCR amplified from the pTT-0012HC plasmid usingPHUSION PCR mix (FinnZymes, cat No. F-531L) and using forward primernumber 572 containing a 5′-end HindIII restriction enzyme site andreverse primer number 686 containing part of hTF.1-219 5′end sequences.The reverse primer 686 was annealed to the hIgG4 hinge region of thepTT-0012HC plasmid and incorporated two point mutations, C239S and C242S(Kabat numbering) in order to remove two free cysteines. The humanTF.1-219 cDNA sequence was PCR amplified using forward primer number 687containing sequence from the 3′end of the 0012V_(H)-CH1-hinge cDNA andreverse primer number 449 containing an EcoRI restriction enzyme sitefor cloning purposes (primer sequences are shown in seq no 70-155). Theresulting 0012V_(H)-CH1-hinge cDNA and hTF.1-219 PCR fragments werecombined and used as template in a second PCR reaction (overlap PCR)using forward primer number 572 and reverse primer number 449. Theresulting PCR fragment encoding 0012V_(H)-CH1-hinge-hTF.1-219 wasdigested with HindIII+EcoRI and inserted into a pTT-based expressionvector resulting in the expression vector designatedpTT-0012V_(H)-CH1-L10-hTF.1-219. The 0012V_(H).T60N and 0012V_(H).T60AcDNAs (including the signal peptide encoding sequence) were excised frompTT-0012HC.T60N or from pTT-0012HC.T60A, respectively using the HinDIIIand NheI restriction enzymes. The resulting variable domains wereinserted into the HinDIII+NheI sites of pTT-0012V_(H)-CH1-L10-hTF.1-219i.e. replacing 0012V_(H) sequence. The resulting expression vectors weredesignated: pTT-0012V_(H).T60N-CH1-L10-hTF.1-219 (also known aspTT-0061V_(H)-CH1-L10-hTF.1-219) andpTT-0012V_(H).T60A-CH1-L10-hTF.1-219 (also known aspTT-0082V_(H)-CH1-L10-hTF.1-219), respectively.

Example 20

Development of pTT-0012V_(H)-CH1-L0-hTF.1-219.

The 0012V_(H)-CH1 encoding DNA sequence was PCR amplified from thepTT-0012HC plasmid using PHUSION PCR mix (FinnZymes, cat No. F-531L) andusing HinDIII containing forward primer number 572 and a reverse primernumber 702 containing DNA sequences overlapping with the 5′end ofhTF.1-219 cDNA sequence. The hTF.1-219 cDNA sequence was PCR amplifiedusing forward primer number 701 containing cDNA sequences overlappingwith the 3′end cDNA sequence for 0012V_(H)-CH1 and reverse primer number449 containing an EcoRI restriction enzyme site for cloning purposes.The resulting PCR fragments were combined and used in a second PCRreaction (overlapping PCR) using forward primer number 572 and reverseprimer number 449. The resulting PCR fragment was digested withHinDIII+EcoRI and inserted into the HinDIII+EcoRI restriction sites of apTT-based expression vector resulting in an expression vector designatedpTT-0012 V_(H)-CH1-no linker-hTF.1-219.

Example 21

Development of pTT-023V_(H)-CH1-L4a-hTF.1-219, pTT-0051HC-L4a-hTF.1-219,pTT-0051V_(H)-CH1-L4a-hTF.1-219, pTT-0052HC-L4a-hTF.1-219 andpTT-0052V_(H)-CH1-L4a-hTF.1-219.

The 0023V_(H), 0051V_(H) and 0052V_(H) cDNA sequences were excised frompTT-023HC, pTT-0051HC or from pTT-0052HC using HinDIII+NheI restrictionenzymes. The resulting DNA fragments were inserted into the HinDIII+NheIrestriction enzyme sites of pTT-0012HC-L4a-hTF.1-219 or ofpTT-0012V_(H)-CH1-L4a-hTF.1-219 i.e. replacing the 0012V_(H) DNAsequence. The resulting expression vectors were designatedpTT-0023V_(H)-CH1-L4a-hTF. 1-219, pTT-0051HC-L4a-hTF.1-219,pTT-0051V_(H)-CH1-L4a-hTF.1-219, pTT-0052HC-L4a-hTF.1-219 andpTT-0052V_(H)-CH1-L4a-hTF.1-219.

Example 22

Development of pTT-0051LC-L4a-hTF.1-219 and pTT-0052LC-L4a-hTF.1-219.

The 0051V_(L) and 0052V_(L) cDNA sequences were excised from pTT-0051LCor from pTT-0052LC using HinDIII+BsiWI restriction enzymes. Theresulting DNA fragments were inserted into the HinDIII+BsiWI restrictionenzyme sites of pTT-0012LC-L4a-hTF.1-219 i.e. replacing the 0012V_(L)DNA sequence. The resulting expression vectors were designatedpTT-0051LC-L4a-hTF.1-219 or pTT-0052LC-L4a-hTF.1-219.

Example 23

Development of pTT-Isotype Control LC-L4a-hTF.1-219, pTT-Isotype ControlLC-HPC4, pTT-Isotype Control HC-L4a-hTF.1-219, pTT-Isotype Control HC,pTT-Isotype Control V_(H)-CH1-HPC4, and pTT-Isotype ControlV_(H)-CH1-L4a-TF.

In order to express a hTF.1-219 fusion proteins based on an isotypecontrol Fab or mAb sequence, V_(H) and V_(L) cDNA sequences wereretrieved based on anti-triNitroPhenyl (ATNP) CDR sequences. The ATNPV_(L) sequence was inserted into the HinDIII+BsiWI restriction enzymesites of the following plasmids: pTT-0012LC, pTT-0012LC-HPC4 andpTT-0012LC-L4a-hTF.1-219 i.e. replacing the 0012V_(L) sequence andresulting in the following expression plasmids: pTT-isotype control-LC,pTT-isotype control-LC-HPC4 and pTT-isotype control-LC-L4a-hTF. 1-219.The ATNP V_(H) sequence was inserted into the HinDIII+NheI restrictionenzyme sites of the following plasmids: pTT-0012HC,pTT-0012V_(H)-CH1-HPC4, pTT-0012V_(H)-CH1-L4a-hTF.1-219 andpTT-0012HC-L4a-hTF.1-219 i.e. replacing the 0012V_(H) sequence andresulting in the following expression plasmids: pTT-isotype control-HC,pTT-isotype control-V_(H)-CH1-HPC4, pTT-isotypecontrol-V_(H)-CH1-L4a-hTF.1-219 and pTT-isotype control-HC-L4a-hTF.1-219.

Example 24

Development of pTT-AP-3LC-17GS-TF.1-219, pTT-AP-3LC.C34S-17GS-TF. 1-219and pTT-AP-3V_(H)-CH1-HPC4.

The AP-3 hybridoma expressing an antiGPIIbIIIa mAb was purchased fromATCC (ATCC Number: HB-242) and the variable domain encoding sequencesfrom AP3 LC and HC were determined. Total RNA was isolated from AP-3hybridoma cells using RNEASY (kit for purifying total RNA from cells,tissues, and yeast) mini kit (Qiagen, cat. no. 74106) and an firststranded cDNA was made using SMART RACE (cDNA amplification kit)(clontech, cat no. PT3269-1), PRIMESCRIPT reverse polymerase (Takara BioInc, code no. 2680A) and employing the 5-CDS primer and SMART IIAoligonucleotide (both included in the SMART RACE kit). The LC and HCvariable domain sequences were PCR amplified using UPM primer mix(included in the SMART RACE kit) together with primer number 69 for LCand UPM primer mix together with primer number 312 (primer sequences areshown in seq no 70-155) for HC. The PCR fragments were cloned into asequencing vector using ZEROBLUNT Topo PCR cloning kit for Sequencing(Invitrogen, cat no K287520) following the instructions of themanufacturer. A potential free Cys at position 34 (according to theKabat numbering system) was identified in the V_(L) cDNA sequence. TheCys residue was mutated to a Ser by employing site-directed mutagenesisusing the QUIKCHANGE Site Directed Mutagenesis Kit (Cat no 200518,Stratagene) and primer number 50 and 51 (primer sequences are shown inseq no 70-155). The resulting V_(L) cDNA sequence was sequenced in orderto verify mutated cDNA sequence.

The AP-3V_(H), AP-3V_(L) and AP-3V_(L).C34S cDNA sequenced was PCRamplified in order to generate expression vectors encodingAP-3V_(H)-CH1-HPC4, AP-3LC-L4a-hTF. 1-219 and AP-3LC.C34S-L4a-hTF.1-219.AP-3V_(H) was PCR amplified using PHUSION PCR mix (FinnZymes, cat No.F-531L) and using forward primer number 842 containing a HinDIIIrestriction enzyme site and reverse primer number 843 containing a NheIrestriction enzyme site for cloning purposes. The AP-3V_(L) andAP-3V_(L).C34S cDNA sequenced were PCR amplified using PHUSION PCR mix(FinnZymes, cat No. F-531L) and using forward primer number 844containing a HinDIII restriction enzyme site and reverse primer number845 (primer sequences are shown in seq no 70-155) containing a BsiWIrestriction enzyme site for cloning purposes. The HinDIII+NheI digestedAP-3V_(H) PCR fragment was inserted into the HinDIII and NheIrestriction enzyme sites of pTT-0012V_(H)-CH1-HPC4 i.e. replacing the0012V_(H) DNA sequence resulting in an expression vector designatedpTT-AP3V_(H)-CH1-HPC4. The HinDIII+BsiWI digested AP-3V_(L) andAP-3V_(L).C34S PCR fragments were inserted into the HinDIII and BsiWIrestriction enzyme sites of pTT-0012LC-L4a-hTF.1-219_i.e. replacing the0012V_(L) DNA sequence and resulting in an expression vector designatedpTT-AP-3LC-L4a-hTF.1-219 and pTT-AP-3LC.C34S-L4a-hTF. 1-219.

Example 25

Development of pTT-0012HC.T60N-His6, pTT-hIgG4-Hinge-CH2-CH3-His6 andpTT-hIgG4-Hinge-CH2-CH3-L4a-hTF.1-219.

In order to develop an expression vector encoding 0012HC.T60N with aC-terminal His-6 tag, site-directed mutagenesis was performed usingQUIKCHANGE lightning kit (GenStar Biosolutions, cat No. T113-01). Inbrief, the site-directed mutagenesis reaction was performed usingpTT-0012HC.T60N as template, forward and reverse primer number 1000 and1001 (primer sequences are shown in seq no 70-155). The primer number1000+1001 annealed to the 3′end of the 0012HC.T60N cDNA sequence andcontained His-6 tag encoding sequences followed by a stop codon. Theresulting plasmid was designated pTT-0012HC.T60N-His6.

In order to develop an expression vector encodinghIgG4-hinge-CH2-CH3-L4a-hTF.1-219 site-directed mutagenesis wasperformed using QUIKCHANGE lightning kit (GenStar Biosolutions, cat No.T113-01) and using pTT-0012HC-L4a-hTF.1-219 as template and forward andreverse primers number 1002 and 1003 (primer sequences are shown in seqno 70-155). The primer numbers 1002+1003 annealed to part of the 0012HCsignal peptide and part of the hIgG4 hinge region and deleted the0012V_(H)-CH1 DNA sequences from pTT-0012HC-L4a-hTF.1-219 plasmid. Theresulting expression vector was designatedpTT-hIgG4-hinge-CH2-CH3-L4a-hTF.1-219. In order to develop an expressionvector encoding hIgG4-hinge-CH2-CH3-His6, site-directed mutagenesis wasperformed using Quichange Lightning kit (Stratagene, cat. no. 200518)and using pTT-hIgG4-hinge-CH2-CH3-L4a-hTF.1-219 as template and forwardand reverse primers number 1000 and 1001. Primer number 1000+1001contained His-6 encoding DNA sequences followed by a stop codon and theyannealed to the 3′-end of the hIgG4 CH3 DNA sequence. The resultingexpression vector was designated pTT-hIgG4-hinge-CH2-CH3-His6.

Example 26

Transient Transfection of HEK293-6E Cells.

All mAb, Fab, and hTF.1-219 fusion proteins were expressed in HEK293-6Esuspension cells by transient transfecting expression plasmids intocells. The individual plasmids combinations underlying the resultingspecific protein compounds are shown in Table 6. HEK293-6E cells weregrown in FREESTYLE HEK293 MEDIUM (animal origin-free, chemicallydefined, protein-free medium) (GIBCO, cat. no. 12338-018) supplementedwith 1% P/S (GIBCO cat. no. 15140-122), 0.1% PLURONIC (clock copolymersbased on ethylene oxide and propylene oxide)(GIBCO, cat. no. 24040-032)and 25 ug/mL GENETICIN (aminoglycoside selective agent) (GIBCO, cat. no.10131-019) and cells were transfected at a cell density of approximately1 mill/mL using 293FECTIN (cationic lipid-based formulation fortransfecting DNA into eukaryotic cells) (Invitrogen, cat. no. 12347-019)according to the instructions of the manufacturer. In brief, for eachliter of HEK293-6E cells, the transfection was performed by diluting atotal of 1 mg of DNA into 30 mL OPTIMEM (reduced-serum media) (dilutionA) and by diluting 1 mL 293FECTIN (cationic lipid-based formulation fortransfecting DNA into eukaryotic cells) into 30 mL OPTIMEM(reduced-serum media) (GIBCO, cat. no. 51985-026, dilution B). DilutionA and B were mixed and incubated at room temperature for 30 minutes. Thetransfection mix was hereafter added to the HEK293-6E cells and cellswere incubated at 37° C. in a humified incubator with orbital rotation(125 rpm). Five to seven days post-transfection, cells were removed bycentrifugation and the resulting cell culture supernatants weresterile-filtrated prior to purification. For all transient transfectionexperiments using co-transfection of 2 expression plasmids, the plasmidswere cotransfected in a 1:1 (ug:ug) plasmid ratio using a total DNAamount of 1 mg for each liter of HEK293-6E cells to be transfected. Forthe expression of protein 0120 and 0121 (Table 6), 3 expression plasmidswere co-transfected into HEK293-6E cells in a 1:1:1 (ug:ug:ug) plasmidratio.

TABLE 6 Protein mAb/Fab or mAb-/Fab-hTF.1-219 fusion ID LC plasmid HCplasmid protein name 0012 pTT-0012LC pTT-0012HC mAb 0012 HC₂; LC₂ 0061pTT-0012LC.C36A pTT-0012HC.T60N mAb 0061 HC₂; LC₂ 0082 pTT-0012LC.C36ApTT-0012HC.T60A mAb 0082 HC₂; LC₂ 0023 pTT-0023LC pTT-0023HC mAb 0023HC₂; LC₂ 0051 pTT-0051LC pTT-0051HC mAb 0051 HC₂; LC₂ 0052 pTT-0052LCpTT-0052HC mAb 0052 HC₂; LC₂ 0062 pTT-0052LC pTT-0052HC.C91Y mAb 0062HC₂; LC₂ 0116 pTT-0012LC pTT-0012HC-L0-hTF.1-219 mAb0012-(HC-L0-hTF.1-219)₂; LC₂ 0086 pTT-0012LC pTT-0012HC-L1-hTF.1-219 mAb0012-(HC-L1-hTF.1-219)₂; LC₂ 0087 pTT-0012LC pTT-0012HC-L2-hTF.1-219 mAb0012-(HC-L2-hTF.1-219)₂; LC₂ 0088 pTT-0012LC pTT-0012HC-L3-hTF.1-219 mAb0012-(HC-L3-hTF.1-219)₂; LC₂ 0018 pTT-0012LC-HPC4pTT-0012HC-L4a-hTF.1-219 mAb 0012-(HC-L4a-hTF.1-219)₂; (LC-HPC4)₂ 0013pTT-0012LC pTT-hTF.1-219-L4b-0012HC mAb 0012-(hTF.1-219-L4b-HC)₂; LC₂0089 pTT-0012LC pTT-0012HC-L5-hTF.1-219 mAb 0012-(HC-L5-hTF.1-219)₂; LC₂0090 pTT-0012LC pTT-0012HC-L6-hTF.1-219 mAb 0012-(HC-L6-hTF.1-219)₂; LC₂0091 pTT-0012LC pTT-0012HC-L7-hTF.1-219 mAb 0012-(HC-L7-hTF.1-219)₂; LC₂0092 pTT-0012LC pTT-0012HC-L8-hTF.1-219 mAb 0012-(HC-L8-hTF.1-219)₂; LC₂0093 pTT-0012LC pTT-0012HC-L9-hTF.1-219 mAb 0012-(HC-L9-hTF.1-219)₂; LC₂0107 pTT-0012LC-L0-hTF.1-219 pTT-0012HC mAb 0012-(LC-L0-hTF.1-219)₂; HC₂0108 pTT-0012LC-L1-hTF.1-219 pTT-0012HC mAb 0012-(LC-L1-hTF.1-219)₂; HC₂0109 pTT-0012LC-L2-hTF.1-219 pTT-0012HC mAb 0012-(LC-L2-hTF.1-219)₂; HC₂0045 pTT-0012LC-L3-hTF.1-219 pTT-0012HC mAb 0012-(LC-L3-hTF.1-219)₂; HC₂0019 pTT-0012LC-L4a-hTF.1-219 pTT-0012HC mAb 0012-(LC-L4a-hTF.1-219)₂;HC₂ 0025 pTT-hTF.1-219-L4b-0012LC pTT-0012HC mAb0012-(hTF.1-219-L4b-LC)₂; HC₂ 0046 pTT-0012LC-L5-hTF.1-219 pTT-0012HCmAb 0012-(LC-L5-hTF.1-219)₂; HC₂ 0047 pTT-0012LC-L6-hTF.1-219 pTT-0012HCmAb 0012-(LC-L6-hTF.1-219)₂; HC₂ 0048 pTT-0012LC-L7-hTF.1-219 pTT-0012HCmAb 0012-(LC-L7-hTF.1-219)₂; HC₂ 0049 pTT-0012LC-L8-hTF.1-219 pTT-0012HCmAb 0012-(LC-L8-hTF.1-219)₂; HC₂ 0050 pTT-0012LC-L9-hTF.1-219 pTT-0012HCmAb 0012-(LC-L9-hTF.1-219)₂; HC₂ 0034 pTT-0023LC-HPC4pTT-0023HC-L4a-hTF.1-219 mAb 0023-(HC-L4a-hTF.1-219)₂; (LC-HPC4)₂ 0035pTT-0023LC-L4a-hTF.1-219 pTT-0023HC mAb 0023-(LC-L4a-hTF.1-219)₂; HC₂0056 pTT-0051LC-HPC4 pTT-0051HC-L4a-hTF.1-219 mAb0051-(HC-L4a-hTF.1-219)₂; (LC-HPC4)₂ 0055 pTT-0051LC-L4a-hTF.1-219pTT-0051HC mAb 0051-(LC-L4a-hTF.1-219)₂; HC₂ 0060 pTT-0052LC-HPC4pTT-0052HC-L4a-hTF.1-219 mAb 0052-(HC-L4a-hTF.1-219)₂; (LC-HPC4)₂ 0059pTT-0052LC-L4a-hTF.1-219 pTT-0052HC mAb 0052-(LC-L4a-hTF.1-219)₂; HC₂0096 pTT-isotype control LC-HPC4 pTT-isotype control HC-L4a-hTF.1-219mAb isotype control-(HC-L4a-hTF.1-219)₂; (LC-HPC4)₂ 0110 pTT-isotypecontrol LC-L4a-hTF.1-219 pTT-isotype control HC mAb isotypecontrol-(LC-L4a-hTF.1-219)₂; HC₂ 0010 pTT-0012LC-HPC4 pTT-0012V_(H)-CH1Fab 0012-LC-HPC4; V_(H)-CH1 0100 pTT-0012LC.C36A pTT-0012V_(H).T60N-CH1Fab 0061 (Fab 0012-LC.C36A; V_(H).T60N-CH1) 0073 pTT-0012LC-HPC4pTT-0012V_(H)-CH1-L0-hTF.1-219 Fab 0012-V_(H)-CH1-L0-hTF.1-219; LC-HPC40011 pTT-0012LC-HPC4 pTT-0012V_(H)-CH1-L4a-hTF.1-219 Fab0012-V_(H)-CH1-L4a-hTF.1-219; LC-HPC4 0014 pTT-0012LC-HPC4pTT-hTF.1-219-L4b-0012V_(H)-CH1 Fab 0012-hTF.1-219-L4b-V_(H)-CH1;LC-HPC4 0057 pTT-0012LC-HPC4 pTT-0012V_(H)-CH1-L10-hTF.1-219 Fab0012-V_(H)-CH1-L10-hTF.1-219; LC-HPC4 0105 pTT-0012LC.C36A-HPC4pTT-0012V_(H).T60N-CH1-L10-hTF.1-219 Fab 0061-V_(H)-CH1-L10-hTF.1-219;LC-HPC4 0106 pTT-0012LC.C36A-HPC4 pTT-0012V_(H).T60A-CH1-L10-hTF.1-219Fab 0082-V_(H)-CH1-L10-hTF.1-219; LC-HPC4 0070 pTT-0012LC-L0-hTF.1-219pTT-0012V_(H)-CH1-HPC4 Fab 0012-LC-L0-hTF.1-219; V_(H)-CH1-HPC4 0071pTT-0012LC-L1-hTF.1-219 pTT-0012V_(H)-CH1-HPC4 Fab 0012-LC-L1-hTF.1-219;V_(H)-CH1-HPC4 0072 pTT-0012LC-L2-hTF.1-219 pTT-0012V_(H)-CH1-HPC4 Fab0012-LC-L2-hTF.1-219; V_(H)-CH1-HPC4 0039 pTT-0012LC-L3-hTF.1-219pTT-0012V_(H)-CH1-HPC4 Fab 0012-LC-L3-hTF.1-219; V_(H)-CH1-HPC4 0020pTT-0012LC-L4a-hTF.1-219 pTT-0012V_(H)-CH1-HPC4 Fab0012-LC-L4a-hTF.1-219; V_(H)-CH1-HPC4 0024 pTT-hTF.1-219-L4b-0012LCpTT-0012V_(H)-CH1-HPC4 Fab 0012-hTF.1-219-L4b-LC; V_(H)-CH1-HPC4 0040pTT-0012LC-L5-hTF.1-219 pTT-0012V_(H)-CH1-HPC4 Fab 0012-LC-L5-hTF.1-219;V_(H)-CH1-HPC4 0041 pTT-0012LC-L6-hTF.1-219 pTT-0012V_(H)-CH1-HPC4 Fab0012-LC-L6-hTF.1-219; V_(H)-CH1-HPC4 0042 pTT-0012LC-L7-hTF.1-219pTT-0012V_(H)-CH1-HPC4 Fab 0012-LC-L7-hTF.1-219; V_(H)-CH1-HPC4 0043pTT-0012LC-L8-hTF.1-219 pTT-0012V_(H)-CH1-HPC4 Fab 0012-LC-L8-hTF.1-219;V_(H)-CH1-HPC4 0044 pTT-0012LC-L9-hTF.1-219 pTT-0012V_(H)-CH1-HPC4 Fab0012-LC-L9-hTF.1-219; V_(H)-CH1-HPC4 0063 pTT-0023LC-L3-hTF.1-219pTT-0023V_(H)-CH1-HPC4 Fab 0023-LC-L3-hTF.1-219; V_(H)-CH1-HPC4 0038pTT-0023LC-L4a-hTF.1-219 pTT-0023V_(H)-CH1-HPC4 Fab0023-LC-L4a-hTF.1-219; V_(H)-CH1-HPC4 0064 pTT-0023LC-L5-hTF.1-219pTT-0023V_(H)-CH1-HPC4 Fab 0023-LC-L5-hTF.1-219; V_(H)-CH1-HPC4 0065pTT-0023LC-L6-hTF.1-219 pTT-0023V_(H)-CH1-HPC4 Fab 0023-LC-L6-hTF.1-219;V_(H)-CH1-HPC4 0066 pTT-0023LC-L7-hTF.1-219 pTT-0023V_(H)-CH1-HPC4 Fab0023-LC-L7-hTF.1-219; V_(H)-CH1-HPC4 0067 pTT-0023LC-L8-hTF.1-219pTT-0023V_(H)-CH1-HPC4 Fab 0023-LC-L8-hTF.1-219; V_(H)-CH1-HPC4 0068pTT-0023LC-L9-hTF.1-219 pTT-0023V_(H)-CH1-HPC4 Fab 0023-LC-L9-hTF.1-219;V_(H)-CH1-HPC4 0033 pTT-0023LC-HPC4 pTT-0023V_(H)-CH1-L4a-hTF.1-219 Fab0023-V_(H)-CH1-L4a-hTF.1-219; LC-HPC4 0053 pTT-0051LC-L4a-hTF.1-219pTT-0051V_(H)-CH1-HPC4 Fab 0051-LC-L4a-hTF.1-219; V_(H)-CH1-HPC4 0054pTT-0051LC-HPC4 pTT-0051V_(H)-CH1-L4a-hTF.1-219 Fab0051-V_(H)-CH1-L4a-hTF.1-219; LC-HPC4 0069 pTT-0052LC-L4a-hTF.1-219pTT-0052V_(H)-CH1-HPC4 Fab 0052-LC-L4a-hTF.1-219; V_(H)-CH1-HPC4 0058pTT-0052LC-HPC4 pTT-0052V_(H)-CH1-L4a-hTF.1-219 Fab0052-V_(H)-CH1-L4a-hTF.1-219; LC-HPC4 0094 pTT-isotype controlLC-L4a-hTF.1-219 pTT-isotype control V_(H)-CH1-HPC4 Fab isotypecontrol-LC-L4a-hTF.1-219; V_(H)-CH1-HPC4 0095 pTT-isotype controlLC-HPC4 pTT-isotype control V_(H)-CH1-L4a-hTF.1-219 Fab isotypecontrol-V_(H)-CH1-L4a-hTF.1-219; LC-HPC4 0128 pTT-AP-3LC-L4a-hTF.1-219pTT-AP-3V_(H)-CH1-HPC4 Fab AP-3-LC-L4a-hTF.1-219; V_(H)-CH1-HPC4 0129pTT-AP-3LC.C34S-L4a-hTF.1-219 pTT-AP-3V_(H)-CH1-HPC4 FabAP-3-LC.C34S-L4a-hTF.1-219; V_(H)-CH1-HPC4 0120 pTT-0012LC.C36ApTT-0012HC.T60N-His6 heterodimer-0061-(0012LC.C36A);pTT-hIgG4-hinge-CH2-CH3-L4a-hTF.1-219 (0012HC.T60N-His6);(hinge-CH2-CH3-L4a-hTF.1-219) 0121 pTT-0012LC.C36A-L4a-hTF.1-219pTT-0012HC.T60N heterodimer-0061-(0012LC.C36A-L4a-pTT-hIgG4-hinge-CH2-CH3-His6 hTF.1-219); (0012HC.T60N);(hinge-CH2-CH3-His6) Note #1: heterodimers designated 0120 and 0121 wasproduced using the 3 plasmid constructs as indicated. Note #2: fusionproteins designated in column four can be compiled from sequences listedin sequence appendix. For example can compound 0011 (Fab0012-VH-CH1-L4a-hTF.1-219; LC-HPC4) be prepared by fusing the IDsequences 51, 61 and 14 for the chain 0012-VH-CH1-L4a-hTF.1-219 and 40and 69 for the chain 0012-LC-HPC4. The resulting protein 0011 is aheterodimer of the 0012-VH-CH1-L4a-hTF.1-219 and the LC-HPC4 polypeptidechains.

Example 27

pcDNA3.1(+)-hTLT-1 ECD-HPC4 Ala Mutant Plasmids

Forty hTLT-1 ECD-HPC4 Ala mutant expression constructs were designedaccording to table 7. The expression constructs were developed byexternal contractor GENEART AG (Im Gewerbepark B35, 93059 Regensburg,Germany) and all 40 expression constructs were made based on theexpression vector designated pcDNA3.1(+). Aliquots of DNA for each ofthe 40 hTLT-1 ECD-HPC4 pcDNA3.1(+) expression construct were transfectedinto HEK293-6E suspension cells in order to transiently express eachhTLT-1 ECD-HPC4 Ala mutant protein (Table 7). Transient transfection andculturing of HEK293-6E cells were performed as described in example Z.

TABLE 7 wt Wild-type 1 L22A 2 V25A 3 Q27A 4 V30A 5 L35A 6 H39A 7 R41A 8L42A 9 Q43A 1 K46A 0 1 Q48A 1 1 F54A 2 1 L55A 3 1 P56A 4 1 E57A 5 1 Q60A6 1 D68A 7 1 R69A 8 1 R70A 9 2 R75A 0 2 L82A 1 2 L86A 2 2 E90A 3 2 M91A4 2 T93A 5 2 Q95A 6 2 E96A 7 2 E97A 8 2 D107A 9 3 R110A 0 3 H116A 1 3R117A 2 3 S119A 3 3 P125A 4 3 E126A 5 3 E128A 6 3 E130A 7 3 S136A 8 3N140A 9 4 K159A 0

Example 28

Purification and Characterisation of Monoclonal Anti-TLT-1 Antibodies.

Purification of the seven recombinantly expressed monoclonal anti-TLT-1antibodies described in table 1 was conducted by a 2-step processcomposed of affinity chromatography using a Protein A MABSELECT SUREresin (GE Healthcare, cat. no. 17-5438-01) and gel filtrationchromatography using a 26/60 SUPERDEX 200 (prep grade gel filtrationmedium) PrepGrade col-umn (GE Healthcare, cat no. 17-1071-01).Purifications were conducted using an ÄktaExplorer chromatography system(GE Healthcare, cat. no. 18-1112-41). The buffer systems used for theaffinity purification step was an equilibration buffer composed of 20 mMNaPhosphate pH 7.2, 150 mM NaCl, an elution buffer composed of 10 mMFormic acid pH 3.5 and an pH-adjustment buffer composed of 0.5 MNaPhosphate pH 9.0. Cell supernatants were applied directly without anyadjustments onto a pre-equilibrated MABSELECT SURE column. The columnwas washed with 15 column volumes of equilibration buffer and themonoclonal antibodies were eluted isocratically in approx. 2-5 columnvolume of elution buffer. The pooled fractions were adjusted to neutralpH using the described pH-adjustment buffer immediately after elution.The protein was further purified and buffer exchanged using said gelfiltration column. The running buffer used for size exclusionchromatography was a 25 mM His pH 6.5, 135 mM NaCl. The flow rate usedwas 2.5 ml/min and the monoclonal anti-TLT1 antibodies eluted as singlepeaks at approx. 0.4 column volumes. Based on analyses of fractions overthe entire peak using the previously described SEC-HPLC method (asdescribed in example 2), pools were prepared which contained pureantibody protein eluting as symmetric peaks at approx. 8.5 min. and witha minimum content of earlier eluting high-molecular weight protein. Thepurified antibodies were characterized using the previously describedSDS-PAGE/Coomassie (as described in example 2) and SEC-HPLC methods,showing that all antibody protein preparations produced were highlyhomogenous. All antibodies displayed expected heavy chain components ofapprox. 50 kDa and light chain components of approx. 25 kDa when usingreducing conditions prior to running the SDS-PAGE/Coomassie analyses.Intact molecular mass determinations were performed using a LiquidChromatography Electrospray Ionisation Time-of-Flight Mass Spectrometrymethod setup on an Agilent 6210 instrument and a desalting columnMassPREP (Waters, cat. no. USRM10008656). The buffer system used was anequilibration buffer composed of 0.1% Formic acid in LC-MS graded-H₂Oand an elution buffer composed of 0.1% Formic acid in LC-MS graded-ACN.All antibodies displayed intact molecular masses of 147.2-148.6 kDa,which is approx. 2.7-3.1 kDa above the theoretical masses of the aminoacid sequences for each of the antibodies. Thus, all the recombinantlyexpressed anti-TLT-1 antibodies displayed post-translationalmodifications corresponding to expected HC N-glycosylations. Finalpurities of 95-99% were obtained for the six antibodies. To verify theN-terminal sequence of the cloned and purified anti-TLT-1 antibodies,EDMAN degradations were performed using an automated sequenator system(Applied Biosystems 494 Protein Sequencer). 10-20 degradation cycleswere conducted for each antibody. Here, expected light and heavy chainsequences were confirmed for the six cloned anti-TLT-1 antibodies. Tomeasure the final protein concentrations, a NANODROP spectrophotometer(Thermo Scientific) was used together with specific extinctioncoefficients for each of the six antibodies ranging from 1.34-1.51.

Example 29

Purification and Characterization of Recombinantly ExpressedFab-hTF.1-219 Proteins.

Purification of the Fab-hTF.1-219 fusion proteins outlined in table 6was conducted using a 2-step process composed of affinity chromatographyusing an anti-HPC4 resin (Roche, cat. no. 11815024001) and a finalbuffer shift. The purification was conducted using an ÄktaExplorerchromatography system (GE Healthcare, cat. no. 18-1112-41). The buffersystems used for the purification step was an equilibration buffercomposed of 20 mM Hepes, pH 7.5, 1.0 mM CaCl2, 100 mM NaCl and 0.005%(v/v) TWEEN (polysorbate surfactant)-80, a wash buffer composed of 20 mMHepes, pH 7.5, 1.0 mM CaCl2, 1.0 M NaCl and 0.005% (v/v) TWEEN(polysorbate surfactant)-80, and an elution buffer composed of 20 mMHepes, pH 7.5, 5.0 mM EDTA and 100 mM NaCl. Cell supernatants wereadjusted with 1 mM CaCl2 final concentration and a pH of 7.5 and appliedonto a pre-equilibrated anti-HPC4 column. The column was washed with 5column volumes of equilibration buffer, 5 column volumes of wash bufferand last with 5 column volumes of equilibration buffer. The Fab-hTF1-219proteins were eluted isocratically in approx. 4 column volumes ofelution buffer. The Fab-hTF1-219 proteins were analyzed usingSDS-PAGE/Coomassie, SEC-HPLC and MALDI-TOF MS analyses as describedpreviously (as described in examples 1 and 28), showing that pure andhomogenous proteins with molecular masses of 78-86 kDa were obtained.Since the theoretical masses of the amino acid sequence for theFab-hTF1-219 constructs were 73-77 kDa, all the expressed proteinscontained post-translational modifications. The proteins were preparedfor assay analyses by either dialyzing into PBS buffer or into a buffercomposed of 25 mM His, 135 mM NaCl, pH 6.5 using a SLIDE-A-LYZERDialysis Cassette 10 kDa MWCO (Pierce, cat. no. 66453) or by using thedesalting resin Sephadex G-25 (GE, cat. no. 17-0033) packed in anappropriate column. To measure final protein concentrations, a NANODROPspectrophotometer (Thermo Scientific) was used together with extinctioncoefficients of 1.31-1.47.

Example 30

Purification and Characterization of Recombinantly ExpressedmAb-hTF.1-219 Proteins.

Purification of mAb-hTF.1-219 fusion proteins described in table 6 wasconducted by a 2-step process composed of affinity chromatography basedeither on a Protein A MABSELECT SURE-resin (GE Healthcare, cat. no.17-5438-01) or an anti-HPC4 resin (Roche, cat. no. 11815024001). Theanti-HPC4 resin resin was used for purification of mAb-hTF.1-219constructs, in which hTF.1-219 was fused C-terminally to the heavychain. These included compounds 0197-0000-0013, 0197-0000-0018,0197-0000-0086, 0197-0000-0087, 0197-0000-0088, 0197-0000-0089,0197-0000-0090, 0197-0000-0091, 0197-0000-0092, 0197-0000-0093,0197-0000-0034, 0197-0000-0056, 0197-0000-0060, 0197-0000-0096, and0197-0000-0116. The remaining mAb-hTF.1-219 fusion proteins described intable 6 were purified using Protein A MABSELECT SURE resin. A gelfiltration chromatography method was used as the final polishpurification. Here a 26/60 SUPERDEX 200 (prep grade gel filtrationmedium) PrepGrade column (GE Healthcare, cat no. 17-1071-01) was used.All purifications were conducted using an ÄktaExplorer chromatographysystem (GE Healthcare, cat. no. 18-1112-41) and based essentially usingthe chromatographic procedures described previously. The mAb-hTF.1-219proteins eluted as single peaks at approx. 0.4 column volumes. Based onanalyses of fractions over the entire peak using the previouslydescribed analytical SEC-HPLC method, pools were prepared which pureprotein which eluted as symmetric peaks at approx. 9 min. with a minimumcontent of earlier eluting high-molecular weight protein.

The purified mAb-hTF.1-219 proteins were characterized using thepreviously described SDS-PAGE/Coomassie and SEC-HPLC methods (asdescribed in example 2), showing that all mAb-hTF.1-219 proteins werehighly pure, i.e. above 90% of non-product related impurities. Intactmolecular mass determinations were performed using the previouslydescribed MALDI-TOF MS method (as described in example 28). AllmAb-hTF.1-219 proteins displayed intact molecular weights of 200-206kDa, which is approx. 8-12 kDa above the theoretical masses of the aminoacid sequence for each of the antibodies. Thus, all mAb-hTF.1-219proteins displayed post-translational modifications. To measure thefinal protein concentrations, a NANODROP spectrophotometer (ThermoScientific) was used together with specific extinction coefficients foreach of the six antibodies ranging from 1.34-1.51.

Example 31

Purification and Characterisation of Heterodimer Protein Designated0120.

Purification of the heterodimer protein designated number 0120 in table6 was conducted as a 4-step process composed of 1) His-affinitychromatography using the Ni-NTA resin (QIAGEN, cat. no. 30430), 2)Buffer change using HIPREP (agarose-based chromatography media) 26/10Desalting column (GE Healthcare, cat. no. 17-5087-01), 3) anion-exchangechromatography using the Q SEPHAROSE HP (GE Healthcare, cat. no.17-1014-03), and 4) size-exclusion chromatography using HiLoad 16/60SUPERDEX 200 (prep grade gel filtration medium) (GE Healthcare, cat. no.17-1069-01). The purifications were conducted using an ÄktaExplorerchromatography system (GE Healthcare, cat. no. 18-1112-41). The buffersystems used for the first purification step was an equilibration buffercomposed of 50 mM Tris, pH7.5, 300 mM NaCl, 10 mM Imidazole, and anelution buffer composed of 50 mM Tris, pH7.5, 300 mM NaCl, 500 mMImidazole. The cell supernatant was adjusted with 10 mM Imidazole finalconcentration and a pH of 7.5 and applied onto a pre-equilibrated Ni-NTAcolumn. The column was washed with 4 column volumes of 2% elutionbuffer. The protein was eluted isocratically in approx. 4 column volumesof 60% elution buffer. The main peak based on UV280 monitoring wascollected and pooled. In the second step, the protein was prepared foranion-exchange chromatograph by shifting into 50 mM Tris, pH7.5 bufferusing a desalting column. The buffer system used for the thirdpurification step was an equilibration buffer composed of 50 mM Tris,pH7.5, and an elution buffer composed of 50 mM Tris, pH7.5, 1M NaCl. Thepool from the second step was directed applied to the pre-equilibrated QSEPHAROSE HP column, washed with 2 column volumes of equilibrationbuffer and eluted in a gradient of 0-100% elution buffer over 10 columnvolumes followed by 3 column volume of 100% elution buffer. The mainpeak was collected and pooled. The buffer used in the fourth step wasPBS. The pool of protein from step 3 was directly applied to HiLoad16/60 SUPERDEX 200 (prep grade gel filtration medium) column. The mainpeak was collected and stored at −80° C. SDS-PAGE/Coomassie 8-15%analysis, SEC-HPLC and LC-MS showed that a pure protein was obtained.One dense protein band was observed from the SDS-PAGE/Coomassie analysiswhich corresponded to said heterodimer protein complex. Reducing theprotein resulted in complete abolishment of the protein complex band,while appearance of three bands indicating three subunits of the proteincomplex. The final protein integrity was analyzed based on a SEC-HPLCmethod set up on a Waters LC 2795/2996 system and using a BIOSEP (columnfor separation biomolecules)-SEC-S3000 300×7.8 mm column (Phenomenex,cat. no. 00H-2146-K0) and a running buffer composed of PBS. The proteinwas eluted as a single symmetric peak at a retention time of approx. 8.2min at a flow rate of 1 ml/min. To measure the final proteinconcentration, a NANODROP spectrophotometer (Thermo Scientific) was usedtogether with an extinction coefficient of 1.34. The molecular weightsof each subunit were determined by LC-MS. Mass deconvolution of the LCsubunit indicated a mass equal to the expected value. Mass deconvolutionof the HC-His subunit indicated a mass equal to the expected value withG0F, G1F and G2F N-glycans. The mass spectrum signal of Fc-sTF was toolow to be deconvoluted due to heavy glycosylation of tissue factor.

Example 32

Purification and Characterisation of Heterodimer Protein Designated0121.

Purification of the heterodimer protein designated number 121 wasessentially the same as described for the heterodimer number 120. Here,the protein was washed in 3 column volumes of equilibration buffer andeluted in 0-40% elution buffer over 8 column volumes followed by 3column volumes of 100% elution buffer. This gradient elution ensured thecomplete separation of the heterodimer with Fc-His homodimer.SDS-PAGE/Coomassie 8-15%, SEC-HPLC and LC-MS showed that a pure proteinwas obtained. One dense protein band was observed on SDS-PAGE/Coomassiewhich corresponded to said heterodimer protein complex. Reducing theprotein resulted in complete abolishment of the protein complex band,while appearance of three bands indicating three subunits of the proteincomplex. The final protein integrity was analyzed based on a SEC-HPLCmethod set up on a Waters LC 2795/2996 system and using a BIOSEP (columnfor separation biomolecules)-SEC-S3000 300×7.8 mm column (Phenomenex,cat. no. 00H-2146-K0) and a running buffer composed of PBS. The proteinwas eluted as a single symmetric peak at a retention time of approx. 8.2min at a flow rate of 1 ml/min. To measure the final proteinconcentration, a NANODROP spectrophotometer (Thermo Scientific) was usedtogether with an extinction coefficient of 1.34. The molecular weight ofeach subunit was determined by LC-MS. Mass deconvolution of the FC-Hissubunit indicated a mass equal to the expected value with G0F, G1F andG2F N-glycans. Mass deconvolution of the HC subunit indicated that theobserved mass correlated with G0F, G1F and G2F N-glycans but with a Lystruncation the C-terminal. The mass spectrum signal of LC-sTF was toolow to be deconvoluted due to heavy glycosylation of tissue factor.

Example 33

Binding of TF-Fusion Proteins to FVIIa.

Binding of TF-fusion proteins to FVIIa was tested by its ability tostimulate FVIIa activity using an amidolytic assay. The effect wascompared with the stimulation induced by soluble TF (sTF), identical tohTF.1-219. Binding of TF to FVIIa results in a marked increase in FVIIacatalytic activity; and binding of sTF and the TF-construct wasconveniently measured using FVIIa's amidolytic activity with thechromogenic substrate S2288 (Ile-Pro-Arg-pNA). Binding of hTF.1-219 toFVIIa increases the FVIIa catalytic activity. The TF-fusion proteinswere designed to locate FVII/FVIIa to the surface of activatedplatelets. Fusion of a protein to TF should therefore not significantlyaffect its binding to FVIIa, and one will expect theconcentration-dependent stimulation of FVIIa activity induced byTF-fusion proteins under optimal conditions to be identical to thatobtained with the non-fused TF (hTF.1-219). In FIG. 7 TF-fusion proteinswere tested in an assay with 50 nM FVIIa, 50 mM Hepes, 0.1 M NaCl, 5 mMCaCl₂, 1 mg/ml BSA pH 7.4 and various concentrations (0-100 nM) of sTF(open square) or Fab-TF-fusion proteins (open symbols) or mAb-TF-fusionproteins (closed symbols). FVIIa amidolytic activity was measured with 1mM of the chromogenic substrate S2288. The FVIIa activity was measuredby the increase in absorbance at 405 nm at room temperature and thereaction was started by addition of 1 mM S2288. Fab-hTF.1-219-fusionproteins stimulate FVIIa amidolytic activity in a concentrationdependent manner indistinguishable from that induced by hTF.1-219showing that these construct bind to FVIIa with a 1:1 stoichiometry andwith a similar affinity as hTF.1-219. The mAb-hTF.1-219-fusion proteinssimilarly stimulate FVIIa activity in a concentration dependent manneridentical to that of hTF.1-219. However, in this case the stoichiometryindicates that these construct bind two FVIIa per mAb as expected withfree access to both TF moieties on the mAb-TF-fusion proteins.

Example 34

mAb Binding and Competition of Different mABs for Binding to TLT-1.

Materials:

TABLE 8 Reagents Reagent Source TLT1 NN mAb0061 NN mAb0023 NN mAb0051 NNmAb0062 NN All other reagents Biacore Human Antibody Capture Kit(BR-1008-39)

Method:

The mAbs of interest were either immobilized directly to a CM5 chip orby capture via a human Fc capture mAb immobilized to a CM5 chip.Reagents that were used are shown in table 8.

Direct Capture:

The TLT-1 mAbs were immobilised to a level of approx 500-1000 RU on aCM5 chip (50 μg/ml diluted in Na-acetate, pH 4.0) using the standardprocedure recommended by the supplier. Two-fold dilutions of TLT1 from200 nM to 0.2 nM were tested for binding to the mABs. Running anddilution buffer: 10 mM HEPES, 150 mM, 0.005% p20, pH 7.4. Regenerationwas obtained by 10 mM Glycine, pH 1.7.

Capture Via Human Fc mAb:

Human Fc mAb was immobilised to approx 10.000 RU. The mAb of interestwas added (approx 100 nM). Two-fold dilutions of TLT1 from 200 nM to 0.2nM were tested. Running and dilution buffer: 10 mM HEPES, 150 mM, 0.005%p20, pH 7.4. Regeneration was obtained in 3 M MgCl₂

Determination of kinetic and binding constants (k_(on), k_(off), K_(D))was obtained assuming a 1:1 interaction of TLT1 and fibrinogen using theBIACORE T100 (instrument for surface plasmon resonance) evaluationsoftware.

Competition:

Competitional binding interaction analysis was obtained by SurfacePlasmon Resonance in a BIACORE T100 (instrument for surface plasmonresonance) analysing binding of various TLT1 mAbs to TLT1 when bound toimmobilised mAb0012 (or an alternative mAb). Direct immobilization to aCM5 chip of the mAbs to a level of 5000-10000 RU was achieved in 10 mMsodium acetate pH 4.5-5.0. This was followed by binding of 50 nM TLT1and after 2 min of dissociation followed by binding of the three othermAbs to be tested for competition. Running and dilution buffer: 10 mMHEPES, 150 mM, 0.005% p20, pH 7.4. Regeneration was obtained by 10 mMGlycine, pH 1.7.

Results:

TABLE 9 TLT1 Biacore binding ka (1/M) kd (1/s) K_(D) (M) TLT1 techniquemAb 0061 9.32E+05 0.003499 3.75E−09 capture mAb 0023 2.87E+05 0.001254.36E−09 direct mAb 0051 2.45E+05 0.00472 1.93E−08 direct mAb 00623.26E+05 0.00134 4.12E−09 direct

TABLE 10 SPR analysis. Binding constant for binding to TLT1. Competitionwith mAB0012 Competition with Competition Competition Competition mAb IDmAB 0012 with 0023 with 0051 with 0062 mAb 0061 yes No no no mAb 0023Yes no yes mAb 0051 yes no mAb 0062 yes

Conclusion:

Binding constants for mAb 0061, 0023, 0051 and 0062 were estimated byBiacore analysis (see table 9).

mAb 0061 and mAB 0051 do not compete with any of the other mAbs forbinding (see table 10). mAb 0023 and mAb 0062 do compete with each other(see table 10).

Example 35

Binding to Activated Platelets by FACS Analysis.

Binding to activated platelets by FACS analysis was shown to bind toboth TLT-1 transfected cells and specifically activated platelets asdescribed below.

Staining with 2F105FabHC-TF fusion protein on platelets using flowcytometry was done by adding platelet preparations (resting versusactivated platelets) in 96 well plates together with 50 μl of diluted2F105FabHC-TF (0011 Fab hTF.1-219) fusion protein or isotype control2F105Fab (0010 Fab) in titration giving final concentrations from 5μl/ml to 0.001 μl/ml. Cell preparations were then incubated at 4 degreesCelsius for 1 hour. After incubation and wash (PBS buffer with 5% Fetalcalf serum, centrifuge for 5 minutes at 200 g) the secondaryRPE-labelled anti-human L+H chain specific antibody (diluted in PBSbuffer 1:100) or a HPC4 specific antibody (specific for tag on 2F105FabHC-TF) was added and incubated for another 1 hour at 4 degrees' Celsius.Finally, cells were washed and fixed by 1% w/v paraformaldehyde) andanalysed in the flow cytometer within 36 hours.

Platelets preparations were produced by making a standard Platelet RichPlasma (PRP). In short, anti-coagulated whole blood was centrifuged (200g for 15 minutes) without brake. The upper layer containing platelets(Platelet rich plasma) were harvested and prostaglandin (Final conc. 5μl/ml) was added for inhibition of platelet activation. Platelets werewashed and used for staining as described above. For production ofactivated platelets a dual agonistic activation was performed, for 10minutes using (62.5 μg/ml Par 1 and Convulxin 100 ng/ml). 50-100.000cells were used pr well.

FIG. 8 shows with two staining procedures that both the isotype Fab andthe 2F105FabHC-TF (0011 Fab hTF.1-219) fusion protein bind specificallyand with high affinity to activated platelets and not to restingplatelets.

Example 36

Enhancement of FVIIa-Mediated of FX Activation by Localization of theFVIIa/TF Complex to the Surface of Pre-Activated Platelets by Binding ofTF-Fusion Proteins to the TLT-1 Receptor.

The capability of TF-fusion proteins to specifically stimulateFVIIa-mediated activation on activated platelets was tested in atwo-step assay in FIG. 9A. The capability of TF-fusion proteins to alsomediate activation of FVII to FVIIa was tested in FIG. 9B. For thispurpose we used lyophilized (activated) platelets from Biopal (REF50710, lot R088001). FX activation was measured in a two step assay inthe absence or presence of platelets at a final concentration of 67.000plt/μl. 100 pM rFVIIa (LASa 15860-008) was mixed with 175 nM FX (EnzymeResearch, Human factor X, HFX 3170 PAL) in 50 mM Hepes, 0.1 M NaCl, 5 mMCaCl₂, 1 mg/ml BSA pH 7.4 at room temperature. FX activation wasarrested after 10 min when an aliquot was removed from each well andadded to an equal volume of ice-cold stopping buffer (50 mM Hepes, 0.1 MNaCl, 20 mM EDTA, 1 mg/ml BSA pH 7.5). The amount of FXa generated inthe samples was then determined in a chromogenic assay by transferring50 μl of the mixture to a microtiter plate well and adding 25 μlCHROMOZYME X (final 0.42 mg/ml) to the well. The absorbance at 405 nmwas measured continuously in a microplate reader (Molecular Devices).FIG. 9A shows the effect of i) 0.03 nM INNOVIN® (lipidated tissuefactor), ii) 10 nM hTF.1-219, iii) 10 nM 0011 Fab-hTF.1-219, iv) 10 nM0010 Fab antibody or v) 0012 mAB antibody on FVIIa-mediated FXactivation in the absence or presence of platelets. FIG. 9B shows theeffect of replacing rFVIIa in the assay with the zymogen, rFVII.

Blockage of FX activation (>95%) was obtained in control experiments bypre-incubating with the goat polyclonal anti-human TF antibody (0.5mg/ml) for 15 min prior to the addition of FVIIa.

The data in FIG. 9A show that fusion of TF with the Fab fragment of anantibody against TLT-1 provides a construct which stimulatesFVIIa-catalyzed activation of FX only in the presence of activatedplatelets. Stimulation of FX activation by the 0011 TF-fusion protein inthe presence of platelets was markedly stronger than stimulation byhTF.1-219. The specificity towards activated platelets is a preferredproperty of the TF-fusion protein, which is not obtained with lipidatedTF (INNOVIN®) that stimulates of FVIIa mediated FX activation also inthe absence of activated platelets.

FIG. 9B shows the effect of replacing rFVIIa in the assay with thezymogen, rFVII. It is well known that INNOVIN® (lipidated tissue factor)stimulates FVIIa-mediated FX activation (FIG. 9A); and also, asindicated by the data in FIG. 9B, that INNOVIN® (lipidated tissuefactor) is capable of stimulating efficient feed-back activation ofFVII. This reaction is essentially independent of the presence ofplatelets. The TF construct, 0011 Fab-hTF.1-219, likewise stimulatesFVIIa-mediated activation of FX and is capable of mediating aplatelet-dependent feed-back activation of FVII (FIG. 9B), however, in anoticeable platelet-dependent manner, and markedly stronger thanhTF.1-219. A significant stimulation of FX activation with the 0010 Fabantibody or 0012 mAb antibody per se was not observed.

Example 37

Enhancement of FVIIa-Mediated of FX Activation by Localization of theFVIIa/TF Complex to the Surface of Activated Platelets by Binding to theTLT-1 Receptor.

In FIG. 10A, 0011 hTF.1-219-fusion protein and hTF.1-219 were tested ina two step assay (see Example 36) in the presence of either resting oractivated platelets at various concentrations. Activated (but notresting) platelets exposed the TLT-1 receptor on their surface.Targeting of a TF fusion protein to the TLT-1 receptor is thought toresult in assembly of the TF/FVIIa complex in a favourable position forstimulation of FX activation on the phospholipid surface of activatedplatelets. FIG. 10B shows the results when the 0011 hTF.1-219-fusionprotein is replaced by 0.1 nM of an AV-hTF.1-219-fusion protein in whichthe TLT-1 antibody is replaced by Annexin V (AV).

Freshly purified, washed resting platelets were prepared from citratestabilized whole blood which was centrifuged 200×g for 10 min. Plateletrich plasma was transferred to 15 ml tubes and supplemented with 1/10volume 2.5% tri-sodium citrate, 1.5% citric acid, 2%. D-glucose.Centrifugation at 500×g/15 min, brake 5. The supernatant was discardedand the platelet pellet dissolved in 10 ml Hepes/Tyrodes buffer (100 mMHepes, 137 mM NaCl, 2.7 mM KCl, 1.7 mM MgCl₂, 5.0 mM D-Glucose, 0.4 mMNaH₂PO₄ pH=6.5) plus 5 μl 10 mg/ml prostaglandin E1. The platelets werecarefully suspended with a plastic pipette and centrifuged at 500×g for15 min, brake 5. The supernatant was discarded and the pelletre-dissolved in 10 ml Hepes/Tyrodes buffer plus 5 μl 10 mg/mlprostaglandin E1. Platelet number was determined by Medonic. Theplatelets rested 30 min or more at room temperature before use.Activation of platelets was obtained by treatment with 5 nM thrombin and100 ng/ml Convulxin.

100 pM rFVIIa was mixed with 10 nM hTF.1-219 or 10 nM 0011 Fab-hTF.1-219fusion protein or 0.1 nM of a AV-hTF.1-219-fusion protein and with 175nM FX (Enzyme Research, Human factor X, HFX 3170 PAL) in 50 mM Hepes,0.1 M NaCl, 5 mM CaCl₂, 1 mg/ml BSA pH 7.4 at room temperature, in thepresence of either resting or activated platelets at variousconcentrations (0-100,000 plt/μl). FX activation was arrested after 10min when an aliquot was removed from each well and added to an equalvolume of ice-cold stopping buffer (50 mM Hepes, 0.1 M NaCl, 20 mM EDTA,1 mg/ml BSA pH 7.5). The amount of FXa generated in the samples was thendetermined in a chromogenic assay by transferring 50 μl of the mixtureto a microtiter plate well and adding 25 μl CHROMOZYME X (final 0.42mg/ml) to the well. The absorbance at 405 nm was measured continuouslyin a microplate reader (Molecular Devices).

The data in FIG. 10A illustrates that binding of the 0011 Fab-hTF.1-219fusion protein to the TLT-1 receptor specifically stimulatedFVIIa-mediated FX activation on activated and not on resting platelets.Stimulation increased with the platelet number and is saturable with anEC₅₀ of about 12,000 activated plts/μl. Targeting to the plateletsurface obtained e.g. by fusion of hTF.1-219 to the TLT-1-binding Fabfragment results in a marked stimulation of FX activation whereas only aminor stimulation is obtained with non-fused hTF.1-219.

FIG. 10B shows the results obtained when 10 nM 0011 Fab-hTF.1-219 fusionprotein is replaced by 0.1 nM of AV-hTF.1-219-fusion protein.AV-hTF.1-219-fusion protein is a more efficient stimulator than 0011Fab-hTF.1-219 of FVIIa-mediated activation of FX on platelets. However,the of AV-hTF.1-219-fusion protein is shown to be less specific towardsactivated platelets than 0011 Fab-hTF.1-219 and a considerablestimulation is induced by the AV-hTF.1-219-fusion protein with restingplatelets. At higher platelet numbers, the stimulation with restingplatelets even exceeds that obtained with activated platelets atequivalent numbers.

Example 38

Effect of FVII/FVIIa and TF-Targeting Constructs Concentrations on theStimulation of FX Activation on Activated Platelets.

A TLT-1-targeting TF-fusion protein with a supposed pro-coagulant effectin haemophilia patients should be able to function under the conditionsprevailing at physiological conditions and to rely on the FVII/FVIIacomposition in the blood. With the assay described in Example 36 it waspossible to examine the stimulatory effect of TF-fusion proteins on theFVIIa-mediated activation of FX on activated platelets at variousconcentrations of FVII/FVIIa (FIG. 11) and at various concentrations ofTF-fusion protein (FIG. 12). Stimulation was obtained in the presence ofadded rFVIIa or rFVII as indicated in the figures.

The results of FIG. 11 show that of FX activation at a fixedconcentration of TF-fusion protein depends on FVII/FVIIa and isstimulated in a concentration dependent manner with maximal stimulationin the physiological relevant range (FVII˜10 nM). The results alsoconfirm that the marked stimulation obtained with a TF-fusion proteinrequires platelet activation and that FVII as well as FVIIa inducestimulation. Furthermore, the results demonstrate the requirement fortargeting by showing that marked stimulation was obtained with fusion ofhTF.1-219 to an anti TLT-1 Fab fragment but not when TF was fused to anequivalent non-targeting Fab fragment.

At a fixed concentration of FVII/FVIIa (10 nM) FIG. 12 examines thestimulation obtained with the 0070 Fab-hTF,1-219 fusion protein as afunction of its concentration. Furthermore FIG. 12 compares this effectwith the stimulation induced by various concentrations of hTF.1-219 or acontrol 0094 isotype Fab-hTF.1-219 fusion protein. The data demonstratethat a marked stimulation was obtained over a wide concentration rangeof the 0070 Fab hTF.1-219 fusion protein, and again demonstrate thesuperiority of a targeting TF-fusion protein relative to thenon-targeting hTF.1-219 and control 0094 isotype Fab-hTF.1-219 fusionproteins. The 0070 Fab hTF.1-219 fusion protein induced a pro-coagulantstimulation over a large concentration interval and was notanti-coagulant at high concentrations. This was in contrast to AV-TFfusion proteins which were observed to be anti-coagulant at highconcentrations (Huang X et al. 2006. Blood 107, 980-986.).

Example 39

Preparation of 20:80 PS:PC Vesicles and Cloning, Expression, Refoldingand Relipidation of TLT-1.

Relipidated TLT-1 in 20:80 PS:PC vesicles were prepared using TRITONX-100 (nonionic surfactant) as detergent as described in Smith andMorrissey (2005) J. Thromb. Haemost., 2, 1155-1162 except that TLT-1 wasused instead of TF.

Materials

LB medium from Sub. Lab. Ba. Kanamycin (50 mg/ml). Kanamycin SigmaK-0254

1000 mM IPTG (IPTG Sigma 1-6758)

Lysis buffer: 1× Bugbuster (Novagen) in 50 mM Tris-HCl, 100 mM NaCl, 2mM EDTA, pH 8.0. Add 0.5 mg/ml lysozyme+DNAseI. Add 1× COMPLETEINHIBITOR COCKTAIL (inhibits proteases)(Roche)

IB-Wash buffer 1: 1:10 bugbuster in IB-buffer. Add 50 μg/mllysozyme+0.5× COMPLETE INHIBITOR COCKTAIL (inhibits proteases) (Roche)

IB-Wash buffer 2: 1:10 Bugbuster in IB-buffer

IB-Buffer: 50 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 8.0

GndHCl buffer: 6M Guanidinium HCl, 50 mM Tris-HCl, 50 mM NaCl, 0.1%TRITON X-100 (nonionic surfactant) red., pH 8.0

Refolding buffer: 50 mM Tris-HCl, 800 mM Arginine, 0.1% TRITON X-100(nonionic surfactant) red., 5 mM reduced glutathione, 0.5 mM oxidizedglutathione pH 8.5 Dialysis buffer: 20 mM Tris-HCl, 0.1% TRITON X-100(nonionic surfactant)_Red., pH 8.0 DTT:Reduced glutathione (Sigma G4251)Oxidized glutathione Sigma G4376 PC:10 mg/ml L-α-phosphatidylcholine(Egg, chicken) in chloroform (Avanti Polar Lipids Inc.) Catalog No.840051C. Mw 760.09

PS: 10 mg/ml L-α-phosphatidylserine sodium salt (Brain, porcine) inchloroform. (Avanti Polar Lipids Inc.) Catalog No. 840032C. Mw 812.05

TRITON X-100 (nonionic surfactant): 10% TRITON X-100 (nonionicsurfactant), hydrogenated, protein grade detergent, sterile filtered.Calbiochem. Catalog No. 648464 Concentration 159 mM (Mw 628)

HBS buffer: 50 mM HEPES, 100 mM NaCl, pH 7.4

Bio-Beads: Bio-Beads SM2 Adsorbent, 20-50 mesh BioRad Laboratories,Catalog No. 152-3920.

Method

Expression: TLT-1 (TLT-1 18-188; SEQ ID NO 182) including extracellulardomain, linker and transmembrane domain was cloned into pET24a usingprimers 1004 (SEQ ID No 183) and 1005 (SEQ ID No 184) and pTT-hTLT-1 astemplate. Standard techniques for DNA preparation were employed.

Transformation was performed into BL21 (DE3). Overnight Culture: 1×50 mlLB medium in 250 ml flasks (plastic) and 50 μl of 50 mg/ml Kanamycin_+1coloni (transformation) from BL21 plate were mixed. The culture wasincubated ON at 37° C., 220 rpm. Starter-culture: 2×500 ml LB medium in2 L flasks (Plastic) with 300 μl of 50 mg/ml Kan was added. 10 ml ONculture TLT-1 lip/pET24a in BL21 (DE3) was added and OD₆₀₀ followed.Incubated at 37° C., 220 rpm

Induction: 2×500 ml with TLT-1 lip/pET24˜BL21 (DE3) in LB. 25° C.˜0.2 mMIPTG was added (100 μl of 1M) to the cell culture when OD₆₀₀ reachedbetween 0.6-0.8. This was incubated for 3 h at 25° C., 220 rpm. Theculture was harvested after 3 h and centrifuged for 30 min at 4600 rpm.The supernatant was discarded. The pellet was stored at −20° C.

Lysis of Inclusion Bodies:

The E. coli pellet was resuspended in 5 ml lysis buffer/g pellet. MgSO4was added to 5 mM to support DNAseI activity. Cell suspension wasincubated on shaking platform for 20 min at room temperature. The lysatewas cleared by centrifugation 20000 g (8500 rpm) for 20 min at 4° C. Thepellet was resuspended in 100 ml IB-Wash buffer. Suspension was mixed bygentle vortexing and incubated at RT for 5 min. Suspension wascentrifuged at 20000 g for 20 min at 4° C. to collect inclusion bodies.Inclusion bodies was resuspended in 100 ml IB-Wash buffer 2. Sample wascentrifuged at 20000 g for 20 min at 4° C. to collect inclusion bodies.The pellet was resuspended in 100 ml water and centrifuged at 20000 gfor 20 min at 4° C. to collect inclusion bodies.

Refolding:

The pellet was resolubilised in x ml GdnHCl buffer (20 ml). The finalconcentration of TLT-1 (A280 was measured) was 1-2 mg/ml. DTT (400 μl)was added to final concentration of 20 mM. Complete solubilization wasensured by magnetic stirring for ˜1-2.5 hrs (1.5 h) at RT. Insolublematerial was removed by centrifugation at 20000 g for 20 min. Aperistaltic pump was used slowly (overnight) to transfer theGdnHCl/protein solution (20 ml) to >20× Refolding buffer (400 ml) at 4°C. The refolding buffer was stirred fast to ensure rapid dilution. Pumprun was obtained at Flow rate 1×, speed 2.5, 4° C. and left overnight at4° C. Precipitated protein was removed by centrifugation at 20000 g(8500 rpm) in 50 ml tubes for 30 min. The TLT1 lip was concentrated from400 ml to 120 ml in Amico-filter 76 mm dia., 10.000 MWCO at 4.5 bar. Theprotein was checked on an SDS-Page by EtOH-precipitation because of theGdnHCl in the sample. 2×500 μl and 2×25 μl was concentrated in 0.5 mltubes with 10.000 MWCO. 50 μl sample+9 vol. ice-cold 99% EtOH (450 μl)was mixed and placed at −20° C. for 10 min. The sample was centrifugedat full speed 13.000 rpm for 5 min. The supernatant was discarded. Thepellet was washed with 450 μl ice-cold 96% EtOH+50 μl MQ. Centrifugeagain. Let dry (EtOH must be totally eliminated before SDS-PAGE). 100 μlwas resuspended 1× sample buffer

PS:PC Preparation and Relipidation:

The exact protocol described in Smith S A & Morrissey J H (2004) “Rapidand efficient incorporation of tissue factor into liposomes”. J. Thromb.Haemost. 2:1155-1162 was followed for relipidation of TLT-1.

Example 40

Establishment of a FX Activation Screening Assay Based on TLT-1 EnrichedPhospholipid Vesicles.

The use of freshly purified platelets as described in Example 37 is wellsuited to demonstrate proof of principle. However, it is not optimalwith activated platelets from individual donors to screen and ranklarger series of TLT-1-targeting TF-fusion proteins. Each plateletpreparation allows a limited number of tests to be performed and is alsosubject to donor to donor variations. An alternative FX activationscreening assay is established using TLT-1 enriched phospholipidvesicles in stead of activated purified platelets. The surface of thevesicles is composed to mimic the phospholipid composition of activatedplatelets.

The feasibility of the FX activation assay to measure stimulation byTLT-1 targeting TF-fusion proteins was tested as shown in FIG. 13 andprovided the basis for the conditions used for screening of theconstructs. 100 pM rFVIIa/rFVII was mixed with various concentrations(0-200 nM) of hTF.1-219 or the 0070 Fab-hTF,1-219 fusion protein or acontrol 0094 isotype Fab-hTF.1-219 fusion protein and with 175 nM FX(Enzyme Research, Human factor X, HFX 3170 PAL) in 50 mM Hepes, 0.1 MNaCl, 5 mM CaCl₂, 1 mg/ml BSA pH 7.4 at room temperature in the presenceof the TLT-1 enriched phospholipid vesicle preparation at 1:2,000dilution. The FX activation was stopped after 10 min when an aliquot wasremoved from each well and added to an equal volume of ice-cold stoppingbuffer (50 mM Hepes, 0.1 M NaCl, 20 mM EDTA, 1 mg/ml BSA pH 7.5). Theamount of FXa generated in the samples was then determined in achromogenic assay by transferring 50 μl of the mixture to a microtiterplate well and adding 25 μl CHROMOZYME X (final 0.42 mg/ml) to the well.The absorbance at 405 nm was measured continuously in a microplatereader (Molecular Devices).

The data in FIG. 13 show an essential mimic of the pattern obtained withactivated platelets, only we observed a TLT-1 unspecific FX activationwith mAb hTF.1-219 fusion proteins at high concentrations of FVII/FVIIa(data not shown). FVII/FVIIa at 0.1 nM with 10 nM Fab TF.1-219-fusionprotein and 0.1 nM FVII/FVIIa with 1.0 nM mAb TF.1-219-fusion proteinwas therefore applied in the screening assay.

Example 41

Screening of TF-Fusion Proteins to Determine Optimal Conditions forFusion with Anti TLT-1-mAbs or Fractions Thereof is Useful for Selectionof Drug Candidates.

The assay with TLT-1 enriched phospholipid vesicles allowed for acomprehensive screening of a large series of TF-fusion proteins. Theliability the data obtained by this screening was also tested bycomparison to data obtained with the FX activation method applied inexample 41. With each preparation of activated platelets the stimulationobtained was compared to the stimulation obtained with the 0020Fab-hTF.1-219 fusion protein set to 100%. The same relative scale wasused for the assay with TLT-1 enriched phospholipid vesicles. Theresults of such screening are compared in FIGS. 27A (mAB fusionproteins) and 27B (Fab fusion proteins).

The data shows that stimulation of FVII/FVIIa-mediated activationtargeted to proteins exposed on activated platelets was obtained with avariety of hTF.1-219 fusion proteins.

The relative efficacy of four different epitopes on TLT-1 to mediate TFtargeting was tested with TF-fusion proteins in which different mAb orFab fragments were fused with identical Linker-hTF.1-219 moieties.Comparison of mAb/Fab 0012, 0023, 0051 and 0052 fusion proteins showsthe following ranking of pro-coagulant potency: 0012>0051>0052=0023.

Additionally FIG. 27A/B allows a ranking of the pro-coagulant potency ofTF fused to a full mAb or to various parts or compositions this mAbwhile still retaining its affinity for the TLT-1 epitope intact.Comparison of identical linker-hTF,1-219 constructs fused to either amAb or a Fab fragment shows that Fab-linker-hTF.1-219-fusion proteinsare superior to mAb-linker-hTF.1-219-fusion proteins. Likewise,Fab-hTF.1-219-fusion proteins are also superior to hetero-dimers inwhich hTF.1-219-fusion proteins are obtained by fusion of hTF.1-219 tothe C-terminal of one of the two HC's or one of the two LC's of the mAb.

Fusion of hTF.1-219 to either the N-terminal or the C-terminal ofantibody heavy- or light chains is also tested by comparison ofotherwise equivalent TF-fusion proteins. The data suggest thatC-terminal fusion is superior to N-terminal fusion.

Comparison of equivalent TF-fusion proteins in which only the linkerbetween TF and the mAb/Fab fragment is varied also provides a ranking oflinkers useful for drug candidate selection.

Platelet receptors other than TLT-1 can also be targeted with TF-fusionproteins to obtain FVII/FVIIa-mediated activation of FX on activatedplatelets. This was shown with the TF-fusion proteins 0128 and 0129produced by fusion of TF with the AP3 Fab fragment The AP3-Fab wasdirected towards the GPIIbIIIa receptor which is present on the surfaceof both activated and resting platelets (Phillips, D. R., and Agin, P.P. (1977) J. Biol. Chem. 252, 2121-226). The 0128 Fab-hTF.1-219 and 0129Fab-hTF.1-219 TF-fusion proteins strongly stimulated the response onactivated platelets. However, the response with resting platelets isabout 20% of the response with activated platelets under conditionswhere, in comparison, the response induced by the TLT-1 targetingTF-fusion protein, 0020 Fab-hTF.1-219, with resting platelets is 8% ofthe response with activated platelets. It is conceivable that thestimulation pattern obtained with TF-fusion proteins is a result ofthree major factors: i) platelet receptor density and relative surfaceexposure on resting and activated platelets, ii) surface exposure ofacidic phospholipids and iii) a minimum of spontaneous plateletactivation during the preparation of washed, resting platelets. Thepresence of GPIIbIIIa receptor on the surface of resting platelets mightaccount for a relatively high activity of the AP3 TF-fusion proteins onresting platelets. Fusion proteins based on antibodies to plateletreceptors of crucial importance to haemostasis like GPIIbIIIa are alsolikely to possess antagonistic properties and serve as anti-coagulantsat high concentrations.

Example 42

TF-Fusion Proteins Promotes Fibrin Clot Formation in Hemophilia-LikeWhole Blood.

Hemophilia-like conditions were obtained by incubation of normalcitrate-stabilized human whole blood (HWB) with 10 μg/ml anti-FVIIIantibody (Sheep anti-Human Factor VIII; Hematologic Technologies Inc)for 30 min at room temp. Spontaneous clotting upon re-calcification ofcitrated HWB was strongly inhibited by the presence of anti-FVIIIantibody. TF-fusion protein was added to HWB to demonstrate thecapability of these proteins to revert haemophilia-like conditionsinduced by addition of anti-FVIII antibody to HWB.

Clot formation was measured by thrombelastography (5000 series TEGanalyzer, Haemoscope Corporation, Niles, Ill., USA). Variousconcentrations (0; 0.02; 0.1; 0.2; 1.0; 10 nM) of 0070 TF-fusionprotein, 0094 TF-fusion protein, or hTF.1-219 were added tohaemophilia-like citrated HWB. Clotting was initiated when 340 μl ofnormal or premixed HWB was transferred to a thrombelastograph cupcontaining 20 μl 0.2 M CaCl₂. The TEG trace was followed continuouslyfor up to 120 min. The following TEG variables were recorded: R time(clotting time i.e. the time from initiation of coagulation until anamplitude of 2 mm was obtained), α-angle (clot development measured asthe angle between the R value and the inflection point of the TEGtrace), K (speed of clot kinetics to reach a certain level of clotstrength, amplitude=20 mm), and MA (maximal amplitude of the TEG tracereflecting the maximal mechanical strength of the clot). The TEG tracesobtained with normal HWB (NWB), “haemophilia” blood, and “haemophilia”blood supplemented with (0; 0.02; 0.1; 0.2; 1.0; 10 nM) of 0070 Fab-hTF.1-219-fusion protein is shown in FIG. 14A. Shown in FIG. 14B are theR-time values obtained for the depicted TEG traces. FIG. 14B, inaddition to R-time values for the 0070 Fab-hTF.1-219-fusion protein,also includes R-time values for equivalent concentrations of hTF.1-219and the non-targeting 0094 Fab isotype hTF.1-219-fusion control protein.All data were obtained from one representative donor.

The 0070 Fab-hTF.1-219-fusion protein was observed to efficientlynormalize clotting of haemophilia-like HWB. The pro-coagulant effect inhaemophilia-like HWB of targeting of TF to TLT-1 is demonstrated withthe 0070 Fab-hTF.1-219-fusion protein by comparison to the effectobtained with the 0094 Fab isotype hTF.1-219 fusion protein in which TFis fused to a non-binding control Fab.

Example 43

TLT1-Fab0043-TF Reduced Tail-Bleeding in FVIII-KO Mice Transfused withHuman Platelets.

The effect of a TLT-1-Fab0043-TF construct was tested in a tail-bleedingmodel in haemophilic mice (FVIII-KO mice) transfused with humanplatelets.

Venous human blood was drawn into acid citrate dextrose (ACD; 1.7 ml/10ml). The blood was incubated with 50 ng/ml PGE₁ for 10 min at roomtemperature (RT), followed by centrifugation (200 g; 10 min). Theplatelet rich plasma (PRP) was collected, and incubated with 50 ng/mlPGE₁ for 10 min at RT, followed by centrifugation at 450 g for 10 min atRT. The plasma was removed and the platelet pellet was resuspended inplasma to a concentration of 1.1-2.8 10⁹ plts/ml.

The mice were anaesthetized with pentobarbital and catheterisized. Themice were pre-treated with 1 nmol/kg TLT1-Fab0043-TF (5 ml/kg; n=7) orATNP-FAb-TF (control; irrelevant FAb-TF0095-construct; n=8) through thecatheter. After 3 minutes human platelets (as PRP) were transfused tothe mice (1.1-2.8×10⁸ platelets/mouse; 5 ml/kg), and after another 2minutes tail bleeding was induced followed by a 30 minutes observationperiod. Blood loss was measured as the amount of lost haemoglobin. Onemin after platelet transfusion, 6.5 and 7.5% of the total plateletpopulation was human in the control and TLT1-FAb-TF treated group,respectively.

TLT1-FAb-TF reduced blood loss significantly from 3956±447 to 1180±489nmol haemoglobin (p<0.01).

Example 44

Analysis of Fibrinogen Binding to TLT1 and Binding Competition BetweenTLT1 mAbs and Fibrinogen.

TLT-1 binds fibrinogen as tested by SPR analysis. Furthermore,simultaneous binding of fibrinogen and each of the four mAbs: mAb 0012,mAb 0023, mAb 0051 and mAb 0062 was tested by SPR analysis in a BIACORET100 instrument (for surface plasmon resonance).

Materials used are shown in table 11.

TABLE 11 Reagent Source TLT1 NN mAb001 NN 2 mAb002 3 mAb005 1 mAb006 2Fibrinogen HCI-0150R Haematologic technologies All other Biacore GEHealthcare reagents

Method:

Human TLT1 was immobilised to a level of approx 1000 RU on a CM5 chip(50 μg/ml diluted in Na-acetate, pH 4.0) using the standard procedurerecommended by the supplier. Four-fold dilutions of human fibrinogenfrom 200 nM to 0.2 nM were tested for binding to the immobilized TLT1.Running and dilution buffer: 10 mM HEPES, 150 mM, 0.005% p20, pH 7.4.Regeneration was obtained by 10 mM Glycine, pH 1.7. Determination ofkinetic and binding constants (k_(on), k_(off), K_(D)) was obtainedassuming a 1:1 interaction of TLT1 and fibrinogen using the Biacore T100evaluation software.

Competition of the different mAbs for binding to TLT1 and fibrinogensimultaneously was tested by immobilisation of each of the mAbs toapproximately 10000-15000 RU at a CM5 chip followed by binding of 50 nMTLT1 followed after 2-3 min dissociation by varying concentrations ofthe mAbs to be tested for competition. Regeneration of the chip wasobtained by 10 mM Glycine, pH 1.7.

Results:

TABLE 12 TLT1 binding to fibrinogen ka (1/M) kd (1/s) K_(D) (M)TLT1-fibrinogen 4171 3.92 X 10⁻⁴ 9.40E−08 binding

TABLE 13 Competition with fibrinogen. The mAb of interest wasimmobilised to a chip. Addition of TLT1 was followed by fibrinogen (asandwich). Competition with mAb ID fibrinogen mAb0012 no mAb0023 yesmAb0051 no mAb0062 yes

Conclusion:

Fibrinogen (HCl-0150R) binds fibrinogen. mAb 0023 and mAb 0062 competewith this binding site. mAb 0012 and mAb 0051 do not compete.

Example 45

Epitope Mapping by Hydrogen Exchange Mass Spectrometry (HX-MS).

The HX-MS technique has been employed to identify the TLT-1 bindingepitopes covered by the four monoclonal antibodies mAb 0023, mAb 0051,mAb 0062 and mAb 0061.

For the mapping experiments hTLT-1.20-125, hTLT-1.16-162 andhTLT-1.126-162 corresponding to SEQ ID NO 5, 6 and 7, respectively, wereused. All proteins were buffer exchanged into PBS pH 7.4 beforeexperiments.

Method: HX-MS Experiments.

Instrumentation and Data Recording

The HX experiments were automated by a Leap robot (H/D-x PAL; LeapTechnologies Inc.) operated by the LeapShell software (Leap TechnologiesInc.), which performed initiation of the deuterium exchange reaction,reaction time control, quench reaction, injection onto the UPLC systemand digestion time control. The Leap robot was equipped with twotemperature controlled stacks maintained at 20° C. for buffer storageand HX reactions and maintained at 2° C. for storage of protein andquench solution, respectively. The Leap robot furthermore contained acooled Trio VS unit (Leap Technologies Inc.) holding the pepsin-, pre-and analytical columns, and the LC tubing and switching valves at 1° C.The switching valves have been upgraded from HPLC to Microbore UHPLCswitch valves (Cheminert, VICI AG). For the inline pepsin digestion, 100μL quenched sample containing 200 pmol TLT-1 was loaded and passed overa PO-ROSZYME®-Immobilized Pepsin Cartridge (2.1×30 mm (AppliedBiosystems)) using a isocratic flow rate of 200 μL/min (0.1% formicacid:CH₃CN 95:5). The resulting peptides were trapped and desalted on aVANGUARD (UPLC column) pre-column BEH C18 1.7 μm (2.1×5 mm (WatersInc.)). Subsequently, the valves were switched to place the pre-columninline with the analytical column, UPLC-BEH C18 1.7 μm (2.1×100 mm(Waters Inc.)), and the peptides separated using a 9 min gradient of15-40% B delivered at 150 μL/min from an AQUITY UPLC system (WatersInc.). The mobile phases consisted of A: 0.1% formic acid and B: 0.1%formic acid in CH₃CN. The ESI MS data, and the separate data dependentMS/MS acquisitions (CID) and elevated energy (MS^(E)) experiments wereacquired in positive ion mode using a Q-Tof Premier MS (Waters Inc.).Leucine-enkephalin was used as the lock mass ([M+H]⁺ ion at m/z556.2771) and data was collected in continuum mode.

Data Analysis

Peptic peptides were identified in separate experiments using standardCID MS/MS or MS^(E) methods (Waters Inc.). MS^(E) data were processedusing BiopharmaLynx 1.2 (version 017). CID data-dependent MS/MSacquisition was analyzed using the Mass-Lynx software and in-houseMASCOT database.

HX-MS raw data files were subjected to continuous lockmass-correction.Data analysis, i.e., centroid determination of deuterated peptides andplotting of in-exchange curves, was performed using HX-Express ((VersionBeta); Weis et al., 3. Am. Soc. Mass Spectrom. 17, 1700 (2006)).

Epitope Mapping of mAb 0023:

Amide hydrogen/deuterium exchange (HX) was initiated by a 30-folddilution of hTLT-1.20-125 in the presence or absence of mAb 0023 intothe corresponding deuterated buffer (i.e. PBS prepared in D₂O, 96% D₂Ofinal, pH 7.4 (uncorrected value)). All HX reactions were carried out at20° C. and contained 4 μM hTLT-1.20-125 in the absence or presence of2.4 μM mAb 0023 thus giving a 1.2 fold molar excess of mAb bindingsites. At appropriate time intervals ranging from 10 sec to 8 hours,aliquots of the HX reaction were quenched by an equal volume of ice-coldquenching buffer (1.35M TCEP) resulting in a final pH of 2.6(uncorrected value).

Epitope Mapping of mAbs 0051 and 0062:

Epitope mapping of mAb 0051 and mAb 0062 were performed in a separateexperiment using hTLT-1.20-125 and carried out similarly to the mappingof mAb 0023 as described above.

Epitope Mapping of mAb 0061:

Epitope mapping of mAb 0061 was performed in two separate experimentsusing either the hTLT-1.16-162 protein or the hTLT-1.126-162 peptide.

Experiments were performed similarly as described above for mAb 0023.However, the pepsin column was placed at room temperature forexperiments using hTLT-1.126-162. This results in an increased pepsindigestion efficacy with minimal additional exchange loss.

Results Epitope Mapping of mAb 0023

The HX time-course of 20 peptides, covering 100% of the primary sequenceof TLT-1, were monitored in the presence and absence mAb 0023 for 10 secto 8 hours.

The observed exchange pattern in the presence or absence of mAb 0023 canbe divided into two different groups: One group of TLT-1 peptidesdisplay an exchange pattern that is unaffected by the binding of mAb0023 and another group of TLT-1 peptides that show protection fromexchange upon mAb 0023 binding. The regions displaying protection uponmAb 0023 binding encompass peptides covering TLT-1 residues 36-51, 79-91and 105-120. By comparing the relative amounts of exchange protectionwithin each peptide the epitope for mAb 0023 can be narrowed to residues36-47, VQCHYRLQDVKA (SEQ ID NO: 174) (50%), 82-87, LGGGLL (SEQ ID NO:175) (30%), 108-115, GARGPQIL (SEQ ID NO: 176) (20%) with the relativeexchange protection for each segment noted in parenthesis. An overviewof the peptide map for the 0023 epitope is shown in FIG. 16.

Epitope Mapping of mAb 0051

The HX time-course of 22 peptides, covering 100% of the primary sequenceof TLT-1, were monitored in the presence and absence mAb 0051 for 10 secto 1000 sec.

The observed exchange pattern in the presence or absence of mAb 0051 canbe divided into two different groups: One group of TLT-1 peptidesdisplay an exchange pattern that is unaffected by the binding of mAb0051 and a group that is affected. The regions displaying protectionupon mAb 0051 binding encompass peptides covering residues 52-66,92-120. By comparing the relative amounts of exchange protection withineach peptide the epitope for mAb 0051 can be narrowed to residues 55-66,LPEGCQPLVSSA (SEQ ID NO: 177) (75%) and 110-120, RGPQILHRVSL (SEQ ID NO:178) (25%) as well as a weak interaction in the 92-105 stretch. Anoverview of the peptide map for the 0051 epitope is shown in FIG. 17.

Epitope Mapping of mAb 0062

The HX time-course of 22 peptides, covering 100% of the primary sequenceof TLT-1, were monitored in the presence and absence mAb 0062 for 10 secto 1000 sec.

The observed exchange pattern in the presence or absence of mAb 0062 canbe divided into two different groups: One group of TLT-1 peptidesdisplay an exchange pattern that is unaffected by the binding of mAb0062 and another group of TLT-1 peptides that show protection. Theregions displaying protection upon mAb 0062 binding encompass peptidescovering residues 36-51 and 105-120. By comparing the relative amountsof exchange protection within each peptide the epitope for mAb 0062 canbe narrowed to 36-47, VQCHYRLQDVKA (SEQ ID NO: 179) (60%) and 110-120,RGPQILHRVSL (SEQ ID NO: 180) (40%). An overview of the peptide map forthe 0062 epitope is shown in FIG. 18.

Epitope Mapping of mAb 0061

The epitope for mAb 0061 was mapped in two separate experiments usingeither the hTLT-1.16-162 protein or the hTLT-1.126-162.

For hTLT-1.16-162 the HX time-course of 19 peptides, covering 85% of theprimary sequence of TLT-1, were monitored in the presence and absencemAb 0061 for 10 sec to 8 hours. Due to an O-glycosylation at residue5148, no information could be recorded beyond residue 141.

The observed exchange pattern in the presence or absence of mAb 0061 canbe divided into two different groups: One group of TLT-1 peptidesdisplay an exchange pattern that is unaffected by the binding of mAb0061 and another group of TLT-1 peptides that show protection fromexchange upon mAb 0061 binding. The regions displaying protection uponmAb 0061 binding encompass peptides covering residues 121-141. However,it is important to note that no information is given in this experimentfor residue 142 and beyond. By comparing the relative amounts ofexchange protection within each peptide the epitope for mAb 0061 can benarrowed to begin at residue 130.

In order to gain full information on the mAb 0061 epitope, the mappingexperiment was repeated using the peptide hTLT-1.126-162. This peptidebinds mAb 0061 with high affinity and it is not modified byglycosylation. Thus it should be able to give HX-MS information for theentire region.

The HX time-course of 12 peptides, covering the entire 126-162 TLT-1region were monitored in the presence and absence mAb 0061 for 10 sec to3000 sec.

All the peptides in this 126-162 region display protection from exchangeupon mAb 0061 binding. By comparing the relative amounts of exchangeprotection within each peptide the epitope for mAb 0061 can be narrowedto be within residues 130-145, ETHKIGSLAENA (SEQ ID NO: 181.) Anoverview of the peptide map for the 0061 epitope is shown in FIG. 19.

Example 46

Production, Characterization and Binding Analyses of hTLT1 ECD-HPC4 AlaMutants.

hTLT-1 ECD-HPC4 Alanine mutant constructs were designed according totable 7. The expression constructs were developed by external contractorGENEART AG (Im Gewerbepark B35, 93059 Regensburg, Germany) and allexpression constructs were made based on the expression vectordesignated pcDNA3.1(+). Aliquots of DNA for each of the 40 hTLT-1ECD-HPC4 pcDNA3.1(+) expression construct were transfected intoHEK293-6E suspension cells in order to transiently express each hTLT-1ECD-HPC4 Ala mutant protein (Table 7). Transient transfection andculturing of HEK293 6e cells were performed as described in example A.

Seven days post-transfection, cells were removed by centrifugation andthe resulting hTLT-1 ECD-HPC4 Ala mutant protein containing supernatantswere sterile-filtrated prior to analyses. The concentration of expressedhTLT-1 ECD-HPC4 Ala mutant protein in the cleared cell supernatant wasdetermined using a combination of RP-HPLC and SDS-PAGE/Coomassieanalyses. These ranged from 4-40 μg/mL containing variable degree ofdimer formation. As described previously for production of hTLT proteinused for immunization experiments, monomer/dimer forms of the expressedprotein were observed for all hTLT ECD-HPC4 Ala mutant constructs. Therelative concentration of monomer/dimer hTLT1 ECD-HPC4 protein wasestimated by SDS-PAGE/Coomassie and an average Mw for each mutantpreparation was calculated.

All binding studies were run at 25° C., and the samples were stored at15° C. in the sample compartment on a ProteOn Analyzer (BioRad) thatmeasures molecular interactions in real time through surface plasmonresonance. The signal (RU, response units) reported by the ProteOn isdirectly correlated to the mass on the individual sensor chip surfacespots.

Anti-hFc Polyclonal antibody was immobilized onto separate flow cells ofa GLM sensor chip using a 1:1 mixture of 0.4 M EDAC[1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride] and 0.1 MSulfo-NHS [N-hydroxysulfosuccinimide]. Each antibody was diluted in 10mM sodium acetate pH 5.0 to a concentration of 50 μg/ml, and wasimmobilized to an individual flow cell at 30 μl/min for 240 s. Theantibodies were immobilized to flow cells A1-A6 (horizontal direction).After immobilization, the active sites on the flow cell were blockedwith 1 M ethanolamine. The final immobilization level of captureantibody typically ranged from approximately 9,000 to 10,000 RU in oneexperiment. Capture of the anti-TLT1 antibodies 0197-0000-0023,0197-0000-0051, 0197-0000-0061 and 0197-0000-0062 was conducted bydiluting to 0.5 μg/ml into HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mMEDTA, 0.05% surfactant P20, pH 7.4) and injected at 30 μl/min for 60 sin vertical direction, creating interspot reference points with onlyanti-human Fc antibodies. The final capture level of test antibodiestypically ranged from approximately 200 to 300 RU in one experiment.Binding of wt or Ala mutant hTLT-1 ECD-HPC4 protein was conducted byinjecting over parallel flow cells in horizontal direction to allow forcomparative analyses of binding to different captured anti-TLT1antibodies relative to binding to the interspot references. Each hTLT-1ECD-HPC4 protein was diluted to 100 nM, based on the calculated averageMw, into HBS-EP buffer and injected at 30 μl/min for 240 s. The GLM chipwas regenerated after each injection cycle of analyte via one 18 sinjection of 1 M Formic acid followed by a 18 s injection of 50 mM NaOHat 100 μl/min. This regeneration step removed the anti-TLT1 antibody andany bound TLT1 from the immobilized capture antibody surface, andallowed for the subsequent binding of the next test sample pair. Theregeneration procedure did not remove the directly immobilizedanti-human-Fc capture antibody from the chip surface.

Data analysis was performed using the ProteOn Manager™ Software. Nosignificant non-specific binding to the interspot control surfaces wasobserved. Binding curves were processed by double referencing(subtraction of interspot control surface signals as well as blankbuffer injections over captured anti-TLT1 antibodies). This allowedcorrection for instrument noise, bulk shift and drift during sampleinjections. Binding signal at 10 s after stop of analyte injection wasnormalized to level of captured anti-TLT1 antibody and presented asbinding relative to wt hTLT-1 ECD-HPC4 protein.

The following Ala mutations displayed a significant decrease of bindingto respective anti-TLT1 compared to wt hTLT-1 ECD-HPC4 protein.0197-0000-0051: F54A <0.4 wt; M91A <0.2 wt; R117A <0.2 wt; S119A <0.6wt. 0197-0000-0062: R41A <0.2 wt; L42A <0.6 wt; Q43A <0.4 wt; F54A <0.6wt; M91A <0.4 wt; R110A <0.2 wt; H116A <0.6 wt. 0197-0000-0023: L42A<0.2 wt; Q43A <0.2 wt; K46A <0.2 wt; M91A <0.4 wt; R110A <0.2 wt. Sincedecreased binding could be observed for the hTLT1 ECD-HPC4 mutant M91Afor all 4 anti-TLT1 antibodies, the residue probably has an importantinfluence on protein stability rather than being part of an actualepitope. 0197-0000-0061 did not show a decreased binding to any of themutated TLT-1 variants tested, indicating that the epitope is notcovered by the mutants introduced in the binding study.

Example 47

Crystal Structures Complexes Between Anti-TLT-1 Fab and TLT-1 StalkPeptides.

Expression of 100 anti-TLT-1 Fab for crystallization: The anti-TLT-1 Fabfragment, Fab0100, comprising the heavy chain corresponding to SEQ ID NO162 and the light chain corresponding SEQ ID NO 163 was expressedtransiently in HEK293 cells according to the generalized procedure.

Purification of 0100 anti-TLT-1 Fab for crystallization: Purification ofsaid Fab was conducted by a two-step process composed of affinitychromatography using the kappaSelect resin (GE Healthcare, cat. no.17-5458-01) and size-exclusion chromatography. The purification wasconducted using an ÄktaExplorer chromatography system (GE Healthcare,cat. no. 18-1112-41). The buffer systems used for the purification stepwas an equilibration buffer composed of 10 mM NaPhosphate, pH 7.5 and150 mM NaCl and an elution buffer composed of 20 mM Formic acid, pH 3.0.The supernatant was adjusted with 1 M NaOH to a pH of 7.5 and appliedonto a pre-equilibrated kappaSelect column. The column was washed with 5column volumes of equilibration buffer and the Fab protein wasisocratically eluted using approximately 5 column volumes of elutionbuffer. The Fab protein was analyzed using SDS-PAGE/Coomassie and LC-MSanalyses, showing that a pure and homogenous protein with an expectedmolecular weight of 46.9 kDa was obtained. To measure the proteinconcentration, a NANODROP spectrophotometer (Thermo Scientific) was usedtogether with an extinction coefficient of 1.31. The final polish of theFab protein was conducted using a size-exclusion column (SUPERDEX 200(prep grade gel filtration medium)).

Preparation of peptides for crystallization: The TLT-1-stalk peptidehTLT-1.126-162 (SEQ ID NO 7) was prepared by solid phase peptidesynthesis. Likewise, a shorter version hTLT-1.129-142 of the stalkpeptide corresponding to SEQ ID NO 8 was prepared.

Preparation, crystallization and structure determination of theFab0100:TLT-1 complexes. Preparation of Fab0100:hTLT-1.126-162: Thecomplex between Fab0100 and hTLT-1.126-162 was prepared by adding twotimes molar excess of hTLT-1.126-162 to a solution of anti-TLT-1 Fabfollowed by isolation of the complex by separating excess hTLT-1.126-162using preparative size exclusion chromatography. Thus, the Fab0100:hTLT-1.126-162 complex was prepared by mixing Fab (1100 μl, 98 μM) andhTLT-1.126-162 (155 μl, 1391 μM), both in PBS buffer (pH 7.4). Thecomplex was subjected to gel filtration using a SUPERDEX 200 (prep gradegel filtration medium) HighLoad 26/60 (GE Healthcare) column eluted withPBS-buffer (pH 7.4) at a flow rate of 1 ml/min. Fractions correspondingto a volume of 3 ml were collected. Fractions containing the desiredFab0100: hTLT-1.126-162 complex were pooled and then concentrated usinga centrifugal filter device (AMICON, 10 kDa cut-off) to a proteinconcentration of 8.6 mg/ml. This preparation was used forcrystallization of the Fab0100:hTLT-1.126-162 complex.

Preparation of Fab0100:hTLT-1.129-142: The complex between theanti-TLT-1 Fab and the shorter stalk peptide (hTLT-1.129-142) wassimilarly prepared with the exceptions that the molar ratio betweenhTLT-1.129-142 and Fab was 1.5:1 and that the gel filtration stop wasomitted due to weaker binding of hTLT-1.129-142 compared to that of thelonger stalk peptide (hTLT-1.126-162).

Crystallization and data collection of Fab0100:hTLT-1.129-142 andFab0100:hTLT-1.126-162 complexes: Fab0100:hTLT-1.129-142 andFab0100:hTLT-1.126-162 complexes were at room temperature crystallizedby the sitting drop method. Fab0100:hTLT-1.129-142 was crystallized byadding to the protein solution, in a 1:2 volume ratio(precipitant:protein), a precipitation solution containing 0.04 Mpotassium dihydrogen phosphate, 16% w/v PEG 8,000 and 20% glycerol,while the Fab0100:hTLT-1.126-162 complex was crystallized by adding tothe protein solution, in a 1:1 volume ratio (precipitant:protein), aprecipitation solution containing 20% w/v PEG 10,000 and 0.10 M Hepes pH7.5. A crystal of the Fab0100:hTLT-1.129-142 complex was flash frozen inliquid N₂ and during data collection kept at 100 K by a cryogenic N₂ gasstream. Crystallographic data were subsequently collected to 2.14 Åresolution using a Rigaku Micro-Max-007 HF rotating anode and a marCCD165 X-ray detector. Space group determination, integration and scalingof the data were made by the XDS software package (Kabsch, W. (1993) J.Appl. Crystallogr. 26, 795-800). Cell parameters of the crystal weredetermined to be 82.10, 64.99, 107.73 Å, 90°, 95.120 and 900, for a, b,c, α, β and γ respectively, and the space group was determined to be C2.R_(sym) for intensities of the data set was calculated to be 6.5%.Coordinates from a Fab model of the PDB-deposited (Berman, H. M. et al.(2000) Nucleic Acids Res. 28, 235-242) 1NGZ structure (Yin, J. et al.PNAS us 100, 856-861) was used for structure determination of theanti-TLT-1 Fab molecule. The 1NGZ Fab model was divided into twodomains, the variable and the constant domains, which then were used asindependent search models in a Molecular replacement run by the PHASERsoftware program (Mccoy, A. J. et al. Acta Crystallographica Section DBiological Crystallography 61, 458-464; Mccoy, A. J. et al. J. Appl.Crystallogr. 40, 658-674) of the CCP4 suite (Bailey, S. (1994) ActaCrystallogr. Sect. D-Biol. Crystallogr. 50, 760-763). The ARP-wARPsoftware package (Evrard, G. X. et al. Acta Crystallographica Section D63, 108-117) was subsequently used for automated model building andphasing. Additional crystallographic refinements, using the REFMAC5software program (Murshudov, G. N. et al. Acta Crystallogr. Sect.D-Biol. Crystallogr. 53, 240-255), followed by computer graphicsinspection of the electron density maps, model corrections and building,using the COOT software program (Emsley, P. et al. Acta Crystallogr.Sect. D-Biol. Crystallogr. 60, 2126-2132), were applied. The procedurewas cycled until no further significant improvements could be made tothe model. Final calculated R- and R-free after 3 cycles of manualintervention and following refinements were 0.185 and 0.245,respectively, and the model showed a root-mean-square deviation (RMSD)from ideal bond lengths of 0.022 Å.

A crystal of the Fab0100:hTLT-1.126-162 complex was transferred to acryo-solution containing 75% of the precipitant solution and 25% ofglycerol. The crystal was allowed to soak for about 15 seconds, thenflash frozen in liquid N₂ and during data collection kept at 100 K by acryogenic N₂ gas stream. Crystallographic data were subsequentlycollected to 1.85 Å resolution at beam-line BL911-3 (Ursby, T. et al.(2004) AIP Conference Proceedings 705, 1241-1246) at MAX-lab, Lund,Sweden. Space group determination, integration and scaling of the datawere made in the XDS software package. Cell parameters for thesynchrotron data were determined to be 82.54, 65.32, 108.05 Å, 90°,95.15° and 900, for a, b, c, α, β and γ, respectively, and space groupwas determined to be C2. R_(sym) for intensities of the data set wascalculated to be 6.7%. The crystal was isomorphous with theFab0100:hTLT-1.129-142 crystals and therefore rigid body refinement ofthe Fab0100:hTLT-1.129-142 complex was used for the original phasing ofthe Fab0100:hTLT-1.126-162 followed by automated model building andphasing using the ARP-wARP software package. Additional crystallographicrefinements, using the REFMAC5 software program, followed by computergraphics inspection of the electron density maps, model corrections andbuilding, using the COOT software program, were applied. The procedurewas cycled until no further significant improvements could be made tothe model. Final calculated R- and R-free after 13 cycles of manualintervention and following refinements were 0.171 and 0.223,respectively, and the model showed a RMSD from ideal bond lengths of0.027 Å (Table 13).

Results

As shown in Tables 14 and 15, AntiTLT-1 effectively binds to the stalkof TLT-1. Using the software program AREAIMOL, of the CCP4 programsuite, the average areas excluded in pair-wise interaction betweenanti-TLT-1 and TLT-1 were calculated to be 764 Å². The average areasexcluded in pair-wise interactions gave for the Fab0100:hTLT-1.126-162complex 656 and 871 Å², for anti-TLT-1 and TLT-1 respectively.

Residues in the TLT-1 peptide (hTLT-1.126-162) making direct contacts tothe anti-TLT-1 Fab in the Fab0100: hTLT-1.126-162 complex is defined asthe epitope and residues in the anti-TLT-1 Fab making direct contacts tohTLT-1.126-162 in the Fab0100: hTLT-1.126-162 complex is defined as theparatope. Epitope and paratope residues were identified by running theCONTACTS software of the CCP4 program suite using a cut-off distance of4.0 Å between the anti-TLT-1 Fab and the TLT-1 molecule. The results ofthe contact calculations for the Fab0100:hTLT-1.126-162 complex of thecrystal structures are shown in Tables 14 and 15. The resulting TLT-1epitope for anti-TLT-1 was found to comprise the following residues ofSEQ ID NO 7): Lys 8 (133), Ile 9 (134), Gly 10 (135), Ser 11 (136), Leu12 (137), Ala 13 (138), Asn 15 (140), Ala 16 (141), Phe 17 (142), Ser 18(143), Asp 19 (144), Pro 20 (145), Ala 21 (142) where numbers inparenthesis refer to the corresponding residues in SEQ ID NO 2 (Tab. 14and 15).

The resulting paratope included residues His 31, Asn 33, Tyr 37, His 39,Tyr 54, Phe 60, Ser 96, Thr 97, Val 99 and Tyr 101 of the Fab0100 lightchain corresponding to SEQ ID NO 163 (Table 14), and residues Val 2, Phe27, Arg 31, Tyr 32, Trp 33, Glu 50, Thr 57, Asn 59, Ser 98, Gly 99, Val100 and Thr 102 of the Fab0100 heavy chain corresponding to SEQ ID NO162 (Table 15). The TLT-1 epitope residues involved in hydrogen-bindingare also indicated in Tables 14 and 15.

TABLE 3 Results from the X-ray model refinement to the observed data ofthe Fab0100:hTLT-1.126-162 complex by the software program refmac5.REMARK  3 REFINEMENT. REMARK  3  PROGRAM  : REFMAC 5.5.0109 REMARK  3 AUTHORS  : MURSHUDOV,VAGIN,DODSON REMARK  3   REMARK  3   REFINEMENTTARGET : MAXIMUM LIKELIHOOD REMARK  3   REMARK  3  DATA USED INREFINEMENT. REMARK  3   RESOLUTION RANGE HIGH (ANGSTROMS) :  1.85 REMARK 3   RESOLUTION RANGE LOW (ANGSTROMS) :  34.18 REMARK  3   DATA CUTOFF   (SIGMA(F)) : NONE REMARK  3   COMPLETENESS FOR RANGE   (%) : 99.89REMARK  3   NUMBER OF REFLECTIONS     : 46512 REMARK  3   REMARK  3  FITTO DATA USED IN REFINEMENT. REMARK  3   CROSS-VALIDATION METHOD    :THROUGHOUT REMARK  3   FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3   R VALUE  (WORKING + TEST SET) : 0.17330 REMARK  3   R VALUE   (WORKING SET) : 0.17070 REMARK  3   FREE R VALUE       : 0.22260REMARK  3   FREE R VALUE TEST SET SIZE (%) : 5.0 REMARK  3   FREE RVALUE TEST SET COUNT  : 2463 REMARK  3   REMARK  3  FIT IN THE HIGHESTRESOLUTION BIN. REMARK  3   TOTAL NUMBER OF BINS USED    :   20 REMARK 3   BIN RESOLUTION RANGE HIGH    :  1.850 REMARK  3   BIN RESOLUTIONRANGE LOW     :  1.898 REMARK  3   REFLECTION IN BIN   (WORKING SET) : 3409 REMARK  3   BIN COMPLETENESS (WORKING+TEST) (%) :  99.81 REMARK  3  BIN R VALUE    (WORKING SET) :  0.266 REMARK  3   BIN FREE R VALUE SETCOUNT    :   195 REMARK  3   BIN FREE R VALUE       :  0.309 REMARK  3  REMARK  3  NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK  3  ALL ATOMS      :  3993 REMARK  3   REMARK  3  B VALUES. REMARK  3  FROM WILSON PLOT    (A**2) : NULL REMARK  3   MEAN B VALUE   (OVERALL,A**2) : 14.967 REMARK  3   OVERALL ANISOTROPIC B VALUE. REMARK  3   B11(A**2) :  −0.06 REMARK  3   B22 (A**2) :   0.23 REMARK  3   B33 (A**2) : −0.24 REMARK  3   B12 (A**2) :   0.00 REMARK  3   B13 (A**2) :  −0.36REMARK  3   B23 (A**2) :   0.00 REMARK  3   REMARK  3  ESTIMATED OVERALLCOORDINATE ERROR. REMARK  3   ESU BASED ON R VALUE              (A): 0.116 REMARK  3   ESU BASED ON FREE R VALUE            (A):  0.123REMARK  3   ESU BASED ON MAXIMUM LIKELIHOOD         (A):  0.084 REMARK 3   ESU FOR B VALUES BASED ON MAXIMUM LIKELIHOOD (A**2):  6.165 REMARK 3   REMARK  3  CORRELATION COEFFICIENTS. REMARK  3   CORRELATIONCOEFFICIENT FO-FC   :  0.963 REMARK  3   CORRELATION COEFFICIENT FO-FCFREE :  0.939 REMARK  3   REMARK  3  RMS DEVIATIONS FROM IDEAL VALUES  COUNT  RMS  WEIGHT REMARK  3   BOND LENGTHS REFINED ATOMS   (A): 3538; 0.027 ; 0.022 REMARK  3   BOND ANGLES REFINED ATOMS (DEGREES):  4833 ;2.132 ; 1.958 REMARK  3   TORSION ANGLES, PERIOD 1  (DEGREES):  473 ;6.972 ; 5.000 REMARK  3   TORSION ANGLES, PERIOD 2  (DEGREES):  137;35.607 ;24.453 REMARK  3   TORSION ANGLES, PERIOD 3  (DEGREES):  583;14.216 ;15.000 REMARK  3   TORSION ANGLES, PERIOD 4  (DEGREES):  14;23.096 ;15.000 REMARK  3   CHIRAL-CENTER RESTRAINTS   (A**3):  552 ;0.180 ; 0.200 REMARK  3   GENERAL PLANES REFINED ATOMS  (A):  2664 ;0.013 ; 0.021 REMARK  3   REMARK  3  ISOTROPIC THERMAL FACTORRESTRAINTS.  COUNT  RMS  WEIGHT REMARK  3   MAIN-CHAIN BOND REFINEDATOMS (A**2): 2262 ; 1.399 ; 1.500 REMARK  3   MAIN-CHAIN ANGLE REFINEDATOMS (A**2): 3679 ; 2.333 ; 2.000 REMARK  3   SIDE-CHAIN BOND REFINEDATOMS (A**2): 1276 ; 3.462 ; 3.000 REMARK  3   SIDE-CHAIN ANGLE REFINEDATOMS (A**2): 1139 ; 5.231 ; 4.500 REMARK  3   REMARK  3  NCS RESTRAINTSSTATISTICS REMARK  3   NUMBER OF NCS GROUPS : NULL REMARK  3   REMARK  3 TWIN DETAILS REMARK  3   NUMBER OF TWIN DOMAINS : NULL REMARK  3  REMARK  3   REMARK  3  TLS DETAILS REMARK  3   NUMBER OF TLS GROUPS :  2REMARK  3   ATOM RECORD CONTAINS RESIDUAL B FACTORS ONLY REMARK  3  REMARK  3  TLS GROUP:  1 REMARK  3   NUMBER OF COMPONENTS GROUP :  3REMARK  3   COMPONENTS   C SSSEQI TO C SSSEQI REMARK  3   RESIDUE RANGE: L  1   L 109 REMARK  3   RESIDUE RANGE : H  1   H 113 REMARK  3  RESIDUE RANGE : P  7   P 21 REMARK  3  ORIGIN FOR THE GROUP (A):−4.1790 48.4400 34.3450 REMARK  3  T TENSOR REMARK  3   T11:  0.1731T22:  0.1937 REMARK  3   T33:  0.1093 T12: −0.0155 REMARK  3   T13:−0.0164 T23: −0.0192 REMARK  3  L TENSOR REMARK  3   L11:  1.9367 L22: 0.4840 REMARK  3   L33:  3.8383 L12: −0.1522 REMARK  3   L13: −1.2215L23: −0.1172 REMARK  3  S TENSOR REMARK  3   S11: 0.0447 S12: −0.2657S13:  0.0758 REMARK  3   S21: 0.0958 S22: −0.0414 S23: −0.0674 REMARK  3  S31: 0.0036 S32:  0.0098 S33: −0.0032 REMARK  3   REMARK  3  TLSGROUP:  2 REMARK  3   NUMBER OF COMPONENTS GROUP : 2 REMARK  3  COMPONENTS  C SSSEQI  TO C SSSEQI REMARK  3   RESIDUE RANGE :  L 114 L 219 REMARK  3   RESIDUE RANGE :  H 116  H 215 REMARK  3   ORIGIN FORTHE GROUP (A): −24.4360 51.7710 5.9920 REMARK  3  T TENSOR REMARK  3  T11: 0.0252 T22: 0.0170 REMARK  3   T33: 0.0735 T12: 0.0161 REMARK  3  T13: 0.0018 T23: 0.0048 REMARK  3  L TENSOR REMARK  3   L11: 2.0324L22: 1.6905 REMARK  3   L33: 0.8461 L12: 0.7328 REMARK  3   L13: 0.0695L23: 0.3337 REMARK  3  S TENSOR REMARK  3   S11: −0.0068 S12:  0.0156S13: 0.0515 REMARK  3   S21: −0.0127 S22: −0.0101 S23: 0.1316 REMARK  3  S31: −0.0077 S32: −0.0763 S33: 0.0168 REMARK  3   REMARK  3   REMARK 3  BULK SOLVENT MODELLING. REMARK  3   METHOD USED : MASK REMARK  3  PARAMETERS FOR MASK CALCULATION REMARK  3   VDW PROBE RADIUS : 1.40REMARK  3   ION PROBE RADIUS : 0.80 REMARK  3   SHRINKAGE RADIUS : 0.80REMARK  3   REMARK  3  OTHER REFINEMENT REMARKS: REMARK  3  HYDROGENSHAVE BEEN ADDED IN THE RIDING POSITIONS REMARK  3  U VALUES  : RESIDUALONLY REMARK  3   LINKR    SG CYS L 139      SG ACYS L 199      SS LINKR   SG CYS H 22       SG ACYS H 96      SS LINKR    SG ACYS H 141      SGACYS H 197      SS CISPEP  1  PTHR L  7  PRO L  8        0.00 CISPEP  2 VAL L   99  PRO L  100       0.00 CISPEP  3  TYR L  145  PRO L  146      0.00 CISPEP  4  PHE H  147  PRO H  148       0.00 CISPEP  5  GLU H 149  PRO H  150       0.00

TABLE 14 hTLT-1.126-162 “P” (SEQ ID NO 7) interactions with the Fab0100light chain (SEQ ID NO 163). A cut-off of 4.0 Å was used. The contactswere identified by the CONTACT computer program of the CCP4 suite. Inthe last column “***” indicates a strong possibility for a hydrogen bondat this contact (distance <3.3 Å) as calculated by CONTACT, “*”indicates a weak possibility (distance >3.3 Å). Blank indicates that theprogram considered there to be no possibility of a hydrogen bond.Hydrogen-bonds are specific between a donor and an acceptor, aretypically strong, and are easily identifiable. hTLT-1.126-162 Anti-TLT-1Possibly Res. Res. # Atom Res. Res. # Atom Distance H- Type and Chainname Type and Chain name [Å] bond Ile 9P CB Tyr 101L OH 3.76 Ile 9P CD1Ser 96L O 3.58 Thr 97L O 3.68 Ile 9P CG2 Val 99L CG2 3.51 Ile 9P C Tyr101L OH 3.95 Gly 10P N Tyr 101L OH 3.06 *** Gly 10P CA Tyr 101L OH 3.48Gly 10P C Tyr 101L OH 3.67 Ser 11P N Tyr 101L OH 3.09 *** Ser 11P CB Ser96L OG 3.89 Tyr 37L CD1 3.94 Ser 96L O 3.37 Ser 11P OG Ser 96L OG 2.88*** Ser 96L CA 3.76 Ser 96L CB 3.05 Ser 96L C 3.33 Ser 96L O 2.45 ***Tyr 101L CE1 3.94 Tyr 101L CZ 3.59 Tyr 101L OH 3.37 * Ser 11P O Tyr 37LCE1 3.39 Tyr 37L CZ 3.66 Tyr 37L OH 3.55 * Leu 12P CG Asn 33L ND2 3.41Tyr 37L OH 3.86 Leu 12P CD1 His 31L CE1 3.73 His 31L NE2 3.38 His 31LCD2 3.63 Tyr 37L CE2 3.65 Asn 33L ND2 3.68 Tyr 37L CZ 3.84 Tyr 37L OH3.51 Leu 12P CD2 His 31L CE1 3.57 His 31L NE2 3.91 Asn 33L ND2 3.39 Phe17P CB Tyr 54L CG 3.79 Tyr 54L CE1 3.70 Tyr 54L CD1 3.45 Phe 17P CG Tyr54L CG 3.98 Tyr 54L CD1 3.63 Phe 17P CD1 Tyr 54L CB 3.74 Phe 17P CE1 His39L ND1 3.88 His 39L CE1 3.32 His 39L NE2 3.60 Phe 17P CZ His 39L CE13.36 His 39L NE2 3.66 Tyr 37L CD1 3.98 Phe 17P CE2 Tyr 37L CD1 3.79 Tyr37L CE1 3.84 Phe 17P O Phe 60L CD1 3.84 Phe 60L CE1 3.31 Asp 19P N Phe60L CE1 3.88 Asp 19P CA Phe 60L CZ 3.80 Asp 19P CB Phe 60L CZ 3.85

TABLE 15 hTLT-1.126-162 “P” (SEQ ID NO 7) interactions with Fab0100heavy chain (SEQ ID NO 162). A cut-off of 4.0 Å was used. The contactswere identified by the CONTACT computer program of the CCP4 suite. Inthe last column “***” indicates a strong possibility for a hydrogen bondat this contact (distance <3.3 Å) as calculated by CONTACT, “*”indicates a weak possibility (distance >3.3 Å). Blank indicates that theprogram considered there to be no possibility of a hydrogen bond.Hydrogen-bonds are specific between a donor and an acceptor, aretypically strong, and are easily identifiable. hTLT-1.126-162 Anti-TLT-1Possibly Res. Res. # Atom Res. Res. # Atom Distance H- Type and Chainname Type and Chain name [Å] bond Lys 8P C Asn 59H OD1 3.95 Lys 8P O Asn59H CG 3.69 Asn 59H ND2 3.78 * Trp 33H CH2 3.86 Asn 59H OD1 2.85 *** Thr57H CG2 3.45 Ile 9P CA Trp 33H CH2 3.91 Glu 50H OE2 3.52 Asn 59H OD13.79 Ile 9P CB Glu 50H OE2 3.81 Ile 9P C Trp 33H CZ3 4.00 Trp 33H CH23.65 Glu 50H OE2 3.62 Trp 33H CZ2 3.87 Ile 9P O Trp 33H CZ2 3.92 Gly 10PN Glu 50H CD 3.40 Glu 50H OE1 3.38 * Trp 33H CZ3 3.55 Trp 33H CH2 3.68Glu 50H OE2 2.77 *** Trp 33H CE3 3.85 Trp 33H CZ2 3.99 Gly 101P CA Glu50H CD 3.79 Glu 50H OE1 3.40 Trp 33H CZ3 3.80 Glu 50H OE2 3.61 Trp 33HCE2 3.75 Trp 33H CD2 3.55 Trp 33H CE3 3.58 Trp 33H CZ2 3.99 Gly 10P CVal 100H CG2 3.82 Gly 10P O Val 100H CG2 3.86 Ser 11P N Val 100H CG23.78 Ser 11P CA Val 100H CG2 3.78 Ala 13P CB Trp 33H CD1 3.60 Trp 33HNE1 3.52 Asn 15P CG Tyr 32H CD1 3.88 Tyr 32H CE1 3.83 Asn 15P ND2 Arg31H C 3.94 Arg 31H O 3.09 *** Tyr 32H CG 3.98 Tyr 32H CD1 3.76 Tyr 32HCE1 3.70 Tyr 32H CZ 3.88 Arg 31H NH1 3.93 * Ala 16P O Val 100H CB 3.86Val 100H CA 3.80 Val 100H N 2.91 *** Thr 102H CG2 3.46 Gly 99H CA 3.74Gly 99H C 3.78 Phe 17P CA Thr 102H CG2 3.64 Phe 17P CD1 Val 100H O 3.93Phe 17P CE1 Val 100H CB 3.46 Val 100H CG1 3.52 Val 100H O 3.86 Phe 17PCZ Val 100H CB 3.81 Val 100H CG1 3.89 Phe 17P C Thr 102H CG2 3.59 Phe17P O Thr 102H CG2 3.79 Ser 18P C Thr 102H CG2 3.94 Ser 18P O Thr 102HCB 3.48 Thr 102H OG1 3.64 * Thr 102H CG2 3.26 Pro 20P CA Tyr 32H OH 3.47Tyr 32H CZ 3.90 Pro 20P CB Val 2H CG2 3.92 Phe 27H CB 3.93 Phe 27H CD13.86 Phe 27H CG 3.78 Pro 20P CG Thr 102H OG1 3.88 Ser 98H OG 3.54 Pro20P CD Thr 102H CB 3.95 Thr 102H OG1 3.83 Pro 20P C Val 2H CG2 3.98 Pro20P O Phe 27H CB 3.52 Ala 21P N Val 2H CG2 3.51 Ala 21P CA Val 2H CG23.97 Ala 21P CB Val 2H CG2 3.77

Example 48

Epitope Mapping by Peptide Walk.

The peptide walking ELISA defined the minimal binding region of thepeptide. This was established by coating biotinylated peptides with oneresidue frameshift in the stalk region of TLT-1 in streptavidin platesfollowed by binding of the antibody of interest (mAb 0061). A secondaryantibody was added for detection and binding was measured at 450 nm.Positive control: binding to biotinylated TLT-1.

Materials

-   10×PBS: 10×GPBS 14200 Gibco-   TWEEN (polysorbate surfactant)20:Aldrich Cat#27,434-8,    Lot#S30950-315-   Plate: 96-well Streptavidin coated plate Nunc#466014-   BSA: A7030-100 g Lot#057K0737-   Blocking/Dilute Buffer: 1×PBS pH=7.4    -   2% BSA    -   0.5% TWEEN (polysorbate surfactant)20-   Wash Buffer: 1×PBS+0.5% TWEEN (polysorbate surfactant)20-   Standard: Biotinylated TLT-1 04/09-08 1 mg/ml-   mAb: 0197-0000-0061-4A-0.55 mg/ml-   Detecting Ab: Goat anti-Human IgG HRP-labeled 1 mg/ml Prod. no.    NEF802001EA-   TMB Substrate: Ready to use Cat#4390L lot.#70904-   Stop Solution: 2M H₃PO₄

Dilution of Biotinylated TLT-1:

1 mg/ml->6.3 ng/ml (158500× Dilution)

Concentration in well: 0.63 ng

Dilution of Biotinylated Peptides:

Approx conc 2-5 mg/ml (2.5 mg/ml)

2.5 mg/ml->10000× Dilution (25 ng/well): 100 μl of each peptide in eachwell.

Dilution of mAb 0061

0.55 mg/ml->100 ng/ml (5500× Dilution)

Concentration in well: 10 ng

Dilution of mAb Goat Anti-Human IgG HRP:

1 mg/ml->0.2 μg/ml (5000× Dilution)

Synthesis of Biotinylated Peptides in 96 Well Format.

The biotinylated peptides were synthesised using standard solid phasepeptide synthesis. Solutions of 0.3M Fmoc-protected amino acids in 0.3 M1-hydroxybenzotriazole (HOBt) in N-methylpyrrolidinone (NMP) werecoupled using diisopropylcarbodiimide (DIC) for 1-4 hours. As solidsupport the Rink amide LL resin (Merck) was used in a 96 microtiterfilterplate (Nunc) and ca. 20 mg resin pr well was used. The synthesiswas performed using the Multipep RS peptide synthesiser from Intavis,Germany and manufacture protocol was used. The removal of Fmoc was doneusing 25% piperidin in NMP. All peptides were coupled with biotin at theN-terminal and 8-amino-3,6-dioxaoctanoic acid was used as a spacerbetween biotin and the peptides. This spacer was also coupled as aFmoc-protected building block according to the synthesis protocol (IRISbiotech, Germany)

Final Deprotection and Workup

The final deprotection was done using 90% trifluoracetic acid (TFA), 5%triisopropylsilane and 5% H2O for 3 hours. A total of 1 ml TFA was usedpr well. The TFA was filtered to 96 deep well (Nunc) and the TFA wasreduced in volume by evaporation to ca. 100-200 ul pr well anddiethylether was added to all wells in order to precipitate thepeptides. The suspension of peptide in diethylether was transferred tosolvent 96 well filter plate (0.47 um, Millipore) and the peptides werewashed twice with diethylether and dried. The peptides were redissolvedin 80% DMSO and 20% water giving a stock solution of ca. 1-3 mg/ml.

Biotinylated 20mer peptides from stalk region of TLT-1 (SEQ ID NO 6)

2-5 mg/ml in 75% DMSO/H2O (biotinylated in N-terminal):

Number of peptide shown at the left:

 2 A 2 2619.8bio-Oeg-L-N-I-L-P-P-E-E-E-E-E-T-H-K-I-G-S-L-A-E  3 A 32620.7bio-Oeg-N-I-L-P-P-E-E-E-E-E-T-H-K-I-G-S-L-A-E-N  4 A 42577.7bio-Oeg-I-L-P-P-E-E-E-E-E-T-H-K-I-G-S-L-A-E-N-A  5 A 52611.7bio-Oeg-L-P-P-E-E-E-E-E-T-H-K-I-G-S-L-A-E-N-A-F  6 A 62585.6bio-Oeg-P-P-E-E-E-E-E-T-H-K-I-G-S-L-A-E-N-A-F-S  7 A 72603.6bio-Oeg-P-E-E-E-E-E-T-H-K-I-G-S-L-A-E-N-A-F-S-D  8 A 82603.6bio-Oeg-E-E-E-E-E-T-H-K-I-G-S-L-A-E-N-A-F-S-D-P  9 A 92545.6bio-Oeg-E-E-E-E-T-H-K-I-G-S-L-A-E-N-A-F-S-D-P-A 10 A102473.6bio-Oeg-E-E-E-T-H-K-I-G-S-L-A-E-N-A-F-S-D-P-A-G 11 A112431.6bio-Oeg-E-E-T-H-K-I-G-S-L-A-E-N-A-F-S-D-P-A-G-S 12 A122373.6bio-Oeg-E-T-H-K-I-G-S-L-A-E-N-A-F-S-D-P-A-G-S-A 13 B 12358.6bio-Oeg-T-H-K-I-G-S-L-A-E-N-A-F-S-D-P-A-G-S-A-N 14 B 22354.6bio-Oeg-H-K-I-G-S-L-A-E-N-A-F-S-D-P-A-G-S-A-N-P 15 B 32330.7bio-Oeg-K-I-G-S-L-A-E-N-A-F-S-D-P-A-G-S-A-N-P-L 16 B 42331.6bio-Oeg-I-G-S-L-A-E-N-A-F-S-D-P-A-G-S-A-N-P-L-E 17 B 52315.5bio-Oeg-G-S-L-A-E-N-A-F-S-D-P-A-G-S-A-N-P-L-E-P 18 B 62345.5bio-Oeg-S-L-A-E-N-A-F-S-D-P-A-G-S-A-N-P-L-E-P-S 19 B 72386.5bio-Oeg-L-A-E-N-A-F-S-D-P-A-G-S-A-N-P-L-E-P-S-Q 20 B 82388.4bio-Oeg-A-E-N-A-F-S-D-P-A-G-S-A-N-P-L-E-P-S-Q-D 21 B 92446.4bio-Oeg-E-N-A-F-S-D-P-A-G-S-A-N-P-L-E-P-S-Q-D-E 22 B102445.5bio-Oeg-N-A-F-S-D-P-A-G-S-A-N-P-L-E-P-S-Q-D-E-K 23 B112418.5bio-Oeg-A-F-S-D-P-A-G-S-A-N-P-L-E-P-S-Q-D-E-K-S 24 B122460.6bio-Oeg-F-S-D-P-A-G-S-A-N-P-L-E-P-S-Q-D-E-K-S-I 25 C 12410.5bio-Oeg-S-D-P-A-G-S-A-N-P-L-E-P-S-Q-D-E-K-S-I-P 26 C 22436.6bio-Oeg-D-P-A-G-S-A-N-P-L-E-P-S-Q-D-E-K-S-I-P-L 27 C 32434.7bio-Oeg-P-A-G-S-A-N-P-L-E-P-S-Q-D-E-K-S-I-P-L-I

Biotinylated 16mer peptides from the stalk region of hTLT-1 (SEQ ID NO6) 2-5 mg/ml in 75% DMSO/H2O:

29 C 5 2219.3 bio-Oeg-L-N-I-L-P-P-E-E-E-E-E-T-H-K-I-G 30 C 6 2193.2bio-Oeg-N-I-L-P-P-E-E-E-E-E-T-H-K-I-G-S 31 C 7 2192.3bio-Oeg-I-L-P-P-E-E-E-E-E-T-H-K-I-G-S-L 32 C 8 2150.2bio-Oeg-L-P-P-E-E-E-E-E-T-H-K-I-G-S-L-A 33 C 9 2166.1bio-Oeg-P-P-E-E-E-E-E-T-H-K-I-G-S-L-A-E 34 C10 2183.1bio-Oeg-P-E-E-E-E-E-T-H-K-I-G-S-L-A-E-N 35 C11 2157.1bio-Oeg-E-E-E-E-E-T-H-K-I-G-S-L-A-E-N-A 36 C12 2175.2bio-Oeg-E-E-E-E-T-H-K-I-G-S-L-A-E-N-A-F 37 D 1 2133.2bio-Oeg-E-E-E-T-H-K-I-G-S-L-A-E-N-A-F-S 38 D 2 2119.2bio-Oeg-E-E-T-H-K-I-G-S-L-A-E-N-A-F-S-D 39 D 3 2087.2bio-Oeg-E-T-H-K-I-G-S-L-A-E-N-A-F-S-D-P 40 D 4 2029.2bio-Oeg-T-H-K-I-G-S-L-A-E-N-A-F-S-D-P-A 41 D 5 1985.2bio-Oeg-H-K-I-G-S-L-A-E-N-A-F-S-D-P-A-G 42 D 6 1935.2bio-Oeg-K-I-G-S-L-A-E-N-A-F-S-D-P-A-G-S 43 D 7 1878.1bio-Oeg-I-G-S-L-A-E-N-A-F-S-D-P-A-G-S-A 44 D 8 1879bio-Oeg-G-S-L-A-E-N-A-F-S-D-P-A-G-S-A-N 45 D 9 1919bio-Oeg-S-L-A-E-N-A-F-S-D-P-A-G-S-A-N-P 46 D10 1945.1bio-Oeg-L-A-E-N-A-F-S-D-P-A-G-S-A-N-P-L 47 D11 1961bio-Oeg-A-E-N-A-F-S-D-P-A-G-S-A-N-P-L-E 48 D12 1987bio-Oeg-E-N-A-F-S-D-P-A-G-S-A-N-P-L-E-P 49 E 1 1945bio-Oeg-N-A-F-S-D-P-A-G-S-A-N-P-L-E-P-S 50 E 2 1959bio-Oeg-A-F-S-D-P-A-G-S-A-N-P-L-E-P-S-Q 51 E 3 2003bio-Oeg-F-S-D-P-A-G-S-A-N-P-L-E-P-S-Q-D 52 E 4 1984.9bio-Oeg-S-D-P-A-G-S-A-N-P-L-E-P-S-Q-D-E 53 E 5 2026bio-Oeg-D-P-A-G-S-A-N-P-L-E-P-S-Q-D-E-K 54 E 6 1998bio-Oeg-P-A-G-S-A-N-P-L-E-P-S-Q-D-E-K-S 55 E 7 2014.1bio-Oeg-A-G-S-A-N-P-L-E-P-S-Q-D-E-K-S-I 56 E 8 2040.1bio-Oeg-G-S-A-N-P-L-E-P-S-Q-D-E-K-S-I-P 57 E 9 2096.2bio-Oeg-S-A-N-P-L-E-P-S-Q-D-E-K-S-I-P-L 58 E10 2122.3bio-Oeg-A-N-P-L-E-P-S-Q-D-E-K-S-I-P-L-I

Method

The epitope mapping consisted of binding of mAb 0061 to two series ofbiotinylated peptides from the stalk region of TLT-1. The biotinylatedpeptides were bound to streptavidin plates.

Stalk Peptide:

(SEQ ID NO: 173) LNILPPEEEEETHKIGSLAENAFSDPAGSANPLEPSQDEKSIPL

1) 20mer peptide mapping with one residue frameshift (20,1) (seematerials)

2) 16mer peptide mapping with one residue frameshift (16,1) (seematerials)

-   1. Plate was pre-washed 3× with 250 μl wash buffer-   2. 100 μl Biotinylated peptide-solution was added (from Masterplate    10000× diluted, one peptide pr well)-   3. Incubated at RT for 1 hour or +5° C. over night-   4. Washed 3× with wash buffer-   5. 100 μl Primary antibody was added (dilution see above)-   6. Incubated at RT for 1 hour-   7. Washed 3× with wash buffer-   8. 100 μl Secondary antibody was added (dilution see above)-   9. Incubated at RT for 1 hour-   10. Washed 3× with wash buffer-   11. 100 μl Substrate/Develop buffer was added (Reaction time 3 min)-   12. 100 μl 2M H3PO4 was added-   13. Endpoint was read at 450 nm

Binding to biotinylated peptide in a well was recorded as “binding” whenthe absorption at 450 nm was above 3. “No binding” was recorded whensignal was below 1. A signal in between was recorded as “weak binding”.

Results

The biotinylated peptides were put into wells in the following way:

Row A: peptide 2-12 (20mers)

Row B: peptide 13-24 (20mers)

Row C: peptide 25-27 (20mers)

Row C: peptide 29-36 (16mers)

Row D: peptide 37-48 (16mers)

Row E: peptide 49-58 (16mers)

Result from Triple Determination:

1 2 3 4 5 6 7 8 9 10 11 12 A <0.1 <0.1 <0.1 >3 >3 >3 >3 >3 >3 >3 >3B >3 >3 >3 >3 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 C <0.1 <0.1 <0.1<0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 >3 D >3 >3 >3 >3 >3 >3 <0.1 <0.1 <0.1<0.1 <0.1 <0.1 E <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 F G H

In summary, the 20mer-peptides (5-16) give rise to strong positivesignals (<3) corresponding to amino acids: IGSLAENAF (SEQ ID NO: 182).The 16mer peptides 36-42 give rise to a strong positive signals (<3)corresponding to KIGSLAENAF (SEQ ID NO: 182).

CONCLUSION

The peptide walking ELISA has defined the minimal binding area of theepitope for binding to mAb 0061 as the following stretch of amino acidresidues: KIGSLAENAF (SEQ ID NO: 182).

This stretch is indeed part of the epitope defined above by the crystalstructure:

(SEQ ID NO: 183) ETHKIGSLAENAFSDP.

1. A fusion protein comprising (i) at least one tissue factorpolypeptide, or biologically functional variant(s) or fragment(s)thereof, which is/are covalently attached to (ii) a ligand that iscapable of binding (iii) a receptor, and/or a fragment or variantthereof, wherein the receptor is only expressed on the surface ofactivated platelets.
 2. The fusion protein according to claim 1, wherein(iii) is TLT-1 or a fragment or variant thereof.
 3. The fusion proteinaccording to claim 1, wherein (ii) is a monoclonal antibody or afragment thereof.
 4. The fusion protein according to claim 3, whereinthe epitope of (ii) comprises one or more residues selected from thegroup consisting of V17, Q18, C19, H20, Y21, R22, L23, Q24, D25, V26,K27, A28, L63, G64, G65, G66, L67, L68, G89, A90, R91, G92, P93, Q94,I95 and L96 of SEQ ID NO:
 5. 5. The fusion protein according to claim 3,wherein the epitope of (ii) comprises one or more residues selected fromthe group consisting of L36, P37, E38, G39, C40, Q41, P42, L43, V44,S45, S46, A47, V73, T74, L75, Q76, E77, E78, D79, A80, G81, E82, Y83,G84, C85, M86, R91, G92, P93, Q94, I95, L96, H97, R98, V99, S110 andL111 of SEQ ID NO:
 5. 6. The fusion protein according to claim 3,wherein the heavy chain of (ii) comprises: a CDR1 sequence of aminoacids 50 to 54 (DYSMH) of SEQ ID NO: 43, wherein one of these aminoacids may be substituted by a different amino acid; and/or a CDR2sequence of amino acids 69 to 85 (VISTYYGDVRYNQKFKG) of SEQ ID NO: 43,wherein one, two, three or four of these amino acids may be substitutedby a different amino acid; and/or a CDR3 sequence of amino acids 118 to129 (APMITTGAWFAY) of SEQ ID NO: 43, wherein one, two or three of theseamino acids may be substituted by a different amino acid.
 7. The fusionprotein according to claim 3, wherein the epitope of (ii) comprises oneor more residues selected from the group consisting of V17, Q18, C19,H20, Y21, R22, L23, Q24, D25, V26, K27, A28, R91, G92, P93, Q94, I95,L96, H97, R98, V99, S100 and L101 of SEQ ID NO:
 5. 8. The fusion proteinaccording to claim 3, wherein the epitope of (ii) comprises one or moreresidues selected from the group consisting of K133, I134, G135, S136,L137, A138, N140, A141, F142, S143, D144, P145 and A146 of SEQ ID NO: 4.9. The fusion protein according to claim 3, wherein the paratope of (ii)comprises one or more residues selected from the group consisting ofH50, N52, Y56, H58, Y73, F79, S115, T116, V118 and Y120 of SEQ ID NO: 40and residues V20, F45, R49, Y50, W51, E68, T75, N77, S116, G117, V118and T120 of SEQ ID NO: 39
 10. The fusion protein according to claim 3,wherein the heavy chain of (ii) comprises: a CDR1 sequence of aminoacids 49 to 53 (RYWMT) of SEQ ID NO: 39, wherein one of these aminoacids may be substituted by a different amino acid; and/or a CDR2sequence of amino acids 68 to 84 (EINPDSSTINYTPSLKD) of SEQ ID NO: 39,wherein one, two, three or four of these amino acids may be substitutedby a different amino acid; and/or a CDR3 sequence of amino acids 117 to121 (GVFTS) of SEQ ID NO: 39, wherein one, two or three of these aminoacids may be substituted by a different amino acid.
 11. A method oftreating coagulopathy comprising administering a fusion proteinaccording to claim
 1. 12. A monoclonal antibody, or fragment thereof,that is capable of binding TLT-1, or a fragment thereof, wherein theepitope of said monoclonal antibody comprises one or more residuesselected from the group consisting of V17, Q18, C19, H20, Y21, R22, L23,Q24, D25, V26, K27, A28, L63, G64, G65, G66, L67, L68, G89, A90, R91,G92, P93, Q94, I95 and L96 of SEQ ID NO:
 5. 13. A monoclonal antibody,or fragment thereof, that is capable of binding TLT-1, or a fragmentthereof, wherein the epitope of said monoclonal antibody comprises oneor more residues selected from the consisting of L36, P37, E38, G39,C40, Q41, P42, L43, V44, S45, S46, A47, V73, T74, L75, Q76, E77, E78,D79, A80, G81, E82, Y83, G84, C85, M86, R91, G92, P93, Q94, I95, L96,H97, R98, V99, S110 and L111 of SEQ ID NO:
 5. 14. A monoclonal antibody,or fragment thereof, that is capable of binding TLT-1, or a fragmentthereof, wherein the epitope of said monoclonal antibody comprises oneor more residues selected from the group consisting of V17, Q18, C19,H20, Y21, R22, L23, Q24, D25, V26, K27, A28, R91, G92, P93, Q94, I95,L96, H97, R98, V99, S100 and L101 of SEQ ID NO:
 5. 15. A monoclonalantibody, or fragment thereof, that is capable of binding TLT-1, or afragment thereof, wherein the epitope of said antibody comprises one ormore residues selected from the group consisting of K133, I134, G135,S136, L137, A138, N140, A141, F142, S143, D144, P145 and A146 of SEQ IDNO:
 4. 16. The fusion protein according to claim 5, wherein the heavychain of (ii) comprises: a CDR1 sequence of amino acids 50 to 54 (DYSMH)of SEQ ID NO: 43, wherein one of these amino acids may be substituted bya different amino acid; and/or a CDR2 sequence of amino acids 69 to 85(VISTYYGDVRYNQKFKG) of SEQ ID NO: 43, wherein one, two, three or four ofthese amino acids may be substituted by a different amino acid; and/or aCDR3 sequence of amino acids 118 to 129 (APMITTGAWFAY) of SEQ ID NO: 43,wherein one, two or three of these amino acids may be substituted by adifferent amino acid.
 17. The fusion protein according to claim 8,wherein the paratope of (ii) comprises one or more residues selectedfrom the group consisting of H50, N52, Y56, H58, Y73, F79, S115, T116,V118 and Y120 of SEQ ID NO: 40 and residues V20, F45, R49, Y50, W51,E68, T75, N77, S116, G117, V118 and T120 of SEQ ID NO:
 39. 18. Thefusion protein according to claim 8, wherein the heavy chain of (ii)comprises: a CDR1 sequence of amino acids 49 to 53 (RYWMT) of SEQ ID NO:39, wherein one of these amino acids may be substituted by a differentamino acid; and/or a CDR2 sequence of amino acids 68 to 84(EINPDSSTINYTPSLKD) of SEQ ID NO: 39, wherein one, two, three or four ofthese amino acids may be substituted by a different amino acid; and/or aCDR3 sequence of amino acids 117 to 121 (GVFTS) of SEQ ID NO: 39,wherein one, two or three of these amino acids may be substituted by adifferent amino acid.
 19. The fusion protein according to claim 9,wherein the heavy chain of (ii) comprises: a CDR1 sequence of aminoacids 49 to 53 (RYWMT) of SEQ ID NO: 39, wherein one of these aminoacids may be substituted by a different amino acid; and/or a CDR2sequence of amino acids 68 to 84 (EINPDSSTINYTPSLKD) of SEQ ID NO: 39,wherein one, two, three or four of these amino acids may be substitutedby a different amino acid; and/or a CDR3 sequence of amino acids 117 to121 (GVFTS) of SEQ ID NO: 39, wherein one, two or three of these aminoacids may be substituted by a different amino acid.